Predictive chromosomal clusters of synchronous and metachronous brain metastases in clear cell renal cell carcinoma

Accepted Manuscript Predictive Chromosomal Clusters of Synchronous and Metachronous Brain Metastases in Clear Cell Renal Cell Carcinoma A. Gutenberg , M.D. Nischwitz , B. Gunawan , C. Enders , K. Jung , M. Bergmann , W. Feiden , R. Egensperger , K. Keyvani , D. Stolke , U. Sure , H.W.S. Schroeder , R. Warzok , R. Schober , J. Meixensberger , W. Paulus , H. Wassmann , W. Stummer , I. Blümcke , M. Buchfelder , F.K.H. van Landeghem , P. Vajkoczy , M. Günther , J. Bedke , A. Giese , V. Rohde , W. Brück , L. Füzesi , B. Sander PII:

S2210-7762(14)00090-8

DOI:

10.1016/j.cancergen.2014.05.004

Reference:

CGEN 287

To appear in:

Cancer Genetics

Received Date: 23 January 2014 Revised Date:

1 May 2014

Accepted Date: 10 May 2014

Please cite this article as: Gutenberg A, Nischwitz M, Gunawan B, Enders C, Jung K, Bergmann M, Feiden W, Egensperger R, Keyvani K, Stolke D, Sure U, Schroeder H, Warzok R, Schober R, Meixensberger J, Paulus W, Wassmann H, Stummer W, Blümcke I, Buchfelder M, van Landeghem F, Vajkoczy P, Günther M, Bedke J, Giese A, Rohde V, Brück W, Füzesi L, Sander B, Predictive Chromosomal Clusters of Synchronous and Metachronous Brain Metastases in Clear Cell Renal Cell Carcinoma, Cancer Genetics (2014), doi: 10.1016/j.cancergen.2014.05.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Predictive Chromosomal Clusters of Synchronous and Metachronous Brain Metastases in Clear Cell Renal Cell Carcinoma

Gutenberg A1,2*, Nischwitz MD3, Gunawan B3, Enders C3, Jung K4, Bergmann M5, Feiden W 6, Egensperger R7, Keyvani K7, Stolke D8, Sure U8, Schroeder HWS9, Warzok R10, Schober R11, Meixensberger J12, Paulus W 13, Wassmann H14, Stummer W 14, Blümcke I15, Buchfelder M16, van

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Landeghem FKH17,18, Vajkoczy P19, Günther M20, Bedke J21, Giese A2, Rohde V1, Brück W 22, Füzesi L3 and Sander B23

Departments of Neurosurgery, 1Georg August University Göttingen, 2Johannes Gutenberg University Mainz, 8University of Duisburg-Essen, 9Ernst Moritz Arndt University Greifswald, Karl Marx University Leipzig, 14Westphalian Wilhelm University Münster, 16Friedrich Alexander

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University Erlangen, 19Charité-University Medicine Berlin, Departments

of

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Gastroenteropathology,

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Georg

August

University

Göttingen,

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Essen,

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Neuropathology Klinikum Bremen-Mitte, University of the Saarland, University of DuisburgErnst Moritz Arndt University Greifswald,

Wilhelm University Münster, Medicine Berlin,

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Karl Marx University Leipzig, 13Westphalian

Friedrich Alexander University Erlangen,

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University of Alberta, Canada,

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Charité-University

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Städtisches Klinikum Brandenburg,

Germany, Georg August University Göttingen, Germany, 4

Institute of Medical Statistics, Georg August University Göttingen, Germany,

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Department of Urology, University of Tübingen, Germany

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Stereology and Electron Microscopy Laboratory, Institute of Clinical Medicine, Faculty of

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Health Sciences, Aarhus University, Denmark

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Address for correspondence:

Angelika Gutenberg, M.D. Department of Neurosurgery University Medical Clinic Mainz Johannes Gutenberg University Mainz, Germany

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Langenbeckstrasse 1 D-55131 Mainz +49-6131-177331

Facsimile:

+49-6131-172774

Email:

[email protected]

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Phone:

Running title: Cytogenetic Evolution of Early and Late Brain Metastasis in Renal Cancer

Key words: clear cell renal cell carcinoma, brain metastasis, comparative genomic hybridization

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(CGH), copy number alterations, tumor progression.

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Conflict of interest statement: The authors declare no conflict of interests.

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Abstract

Here, synchronous (early) and metachronous (late) brain metastasis (BM) events of sporadic ccRCC (n = 148) were retrospectively analyzed using comparative genomic hybridization (CGH). Using oncogenetic tree models and cluster analyses, chromosomal imbalances related to recurrence-free survival (RFS-BM) were observed. Losses at 9p and 9q appeared to be hallmarks of metachronous BM events, whereas an absence of detectable chromosomal changes at 3p was often associated with synchronous BM events. Correspondingly, k-means

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clustering showed that Cluster 1 cases generally exhibited low copy number chromosomal changes that did not involve 3p. Cluster 2 cases had a high occurrence of -9p/-9q (94–98%) deletions, while Cluster 3 cases had a higher frequency of copy number changes, including loss at chromosome 14 (80%). The higher number of synchronous cases in Cluster 1 was also

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associated with a significantly shorter RFS-BM compared to Clusters 2 and 3 (p = 0.02). Conversely, a significantly longer RFS-BM was observed for Cluster 2 versus Clusters 1 and 3 (p = 0.02). Taken together, these data suggest that metachronous BM events of ccRCC are

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characterized by loss of chromosome 9, whereas synchronous BM events may form

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independently of detectable genetic changes at chromosomes 9 and 3p.

ACCEPTED MANUSCRIPT Introduction Renal cell carcinoma (RCC) constitutes only 2% of all cancers [1], yet accounts for 7–10% of brain metastasis (BM) cases [2, 3]. Moreover, following treatment of localized RCC, approximately 20–30% of patients have been reported to develop distant metastasis [4]. Depending on the extent of the primary disease, BM has developed in 2% of patients that exhibited exclusive abdominal spread, and in up to 16% of patients with concurrent pulmonary

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and bone metastasis of RCC [5]. Once BM is evident, the effectiveness of any palliative systemic therapy is very restricted due to the increased risk of side effects such as cerebral hemorrhage [5-12]. In fact, BM with RCC is an independent negative prognostic factor for survival [13]. It has been reported that the mean survival time for patients with BM from RCC is

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three months if the BM is untreated. The survival period increases to 15.5 months if brain surgery and whole brain radiotherapy are applied [14]. However, the onset of BM is unpredictable, with the interval between a primary diagnosis of RCC and BM ranging from a

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concomitant diagnosis to more than 20 years [15-18].

Metastasis to distant sites in RCC is believed to be a highly selective, non-random process comprising a series of sequential genetic events [19, 20]. Histologically and genetically, RCC is a heterogeneous disease that can be subdivided into clear cell (ccRCC), papillary, chromophobe, and mixed cell variants [19, 20]. ccRCC is the most frequent type (70-78%) [21, 22]. Data from genome-wide genetic analyses, including classical cytogenetics, comparative

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genomic hybridization (CGH), and cytogenomic arrays, suggest that losses at 3p and 14q, as well as gains at 5q, may be critical aberrations in the development of ccRCC [23-27]. Furthermore, chromosomal losses at 4, 9, 13q, 14q, and 18, and chromosomal gains at 1q, 7, and 17, have been associated with more aggressive variants of ccRCC [25, 27-32].

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Unfortunately, data regarding chromosomal changes in BM events of RCC are rare, and are currently restricted to case reports. To date, four cases [33] and another single case [29] have

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been reported. In contrast, no information regarding histomorphological subtypes of RCC have been published.

The aim of this study was to analyze chromosomal imbalances in 148 cases of BM of ccRCC as identified by CGH with respect to clinical follow-up data of recurrence and survival. Probabilistic oncogenetic models were subsequently applied to these data. These models allowed multiple cytogenetic pathways to occur simultaneously within a given data set, without assuming a unique order of events [34].

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Materials and Methods

Patients and clinical data Ethics committee approval was obtained for all cases included in this study. The data set included 148 cases of ccRCC BM that were treated at eleven different neuropathology institutions of the Universities of Berlin, Brandenburg, Bremen, Erlangen, Essen, Göttingen, Greifswald, Homburg, Leipzig, Münster, and Bremen in Germany. For these cases, CGH was

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retrospectively performed using formalin-fixed, paraffin-embedded BM tissue samples. In addition, all cases exhibiting ccRCC histomorphology were re-evaluated by an expert pathologist (L.F.). Recurrence-free survival following diagnosis and surgery for ccRCC with BM (RFS-BM) was also evaluated. Cases were categorized as synchronous or metachronous BM,

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and were defined according to the presence or absence of BM at the time of the initial diagnosis. For this series, no BM events occurred within 2–6 months of diagnosis. Overall

Comparative genomic hybridization (CGH)

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survival (OS) was calculated from the initial diagnosis of the primary tumor to death.

DNA Preparation and CGH. Tumor DNA was extracted from formalin-fixed, paraffin-embedded BM samples using proteinase K digestion (2 mg/ml final concentration; Roche, Mannheim, Germany) followed by spin column purification (Qiagen, Hilden, Germany). As a reference, pooled normal DNA from the opposite gender was used as an internal quality control. Labeling

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of tumor DNA with nick translation was performed using biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) as a normal DNA reference. Denatured DNA probes containing 2 µg tumor DNA, 1.5 µg reference DNA, and 80 µg COT-1 DNA were hybridized to normal metaphase spreads on glass slides (15 x 15 mm cover glass area) for 3

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days. The slides were subsequently washed, blocked with bovine serum albumin solution, and incubated with fluorescein-conjugated avidin (Vector Laboratories, Burlingame, CA, USA) and

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rhodamine-conjugated antidigoxigenin (Roche). Finally, the slides were washed and mounted in antifade solution (Vector Laboratories) containing 2.5 µg/ml 4’,6-diamidino-2-phenylindole (DAPI) counterstain.

Imaging and Image Analysis: Image acquisition was performed using a Zeiss Axioskop fluorescence microscope (Zeiss, Göttingen, Germany) equipped with three separate bandpass filters (DAPI bandpass, green single bandpass, and a red single bandpass) and a high sensitivity monochrome charge coupled device (CCD) camera (Photometrics, Tucson, AZ, USA). For each analysis, the mean chromosome-specific green-to-red fluorescence ratios and associated 95% confidence intervals (CI) from at least ten well-selected metaphases were plotted using Quips CGH software (distributed by Applied Imaging, Newcastle, UK). In cases where the baseline value representing the mean green-to-red ratio for the average copy number

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in the entire tumor specimen was 1.0, relative copy number changes were interpreted as gains when the average ratio changes exceeded 1.2, and as losses when the ratio changes were less than 0.8. Alternatively, in highly aneuploid cases, or cases with substantial normal cell contamination, relative copy number changes were classified as gains or losses when the ratio changes varied beyond the 95% CI of the baseline mean green-to-red ratio for the average copy number in the entire tumor specimen. The chromosomal regions 1p32-pter, 13p, 14p, 15p, 19, 21p, and 22p, known constitutive heterochromatic regions at 1q, 9q, 16q, and Yq, and

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telomeric regions were excluded from analysis.

Statistical Methods

For statistical analysis, the detected CGH losses (denoted by ‘-’) and gains (denoted by ‘+‘)

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were grouped according to chromosome arm. P-values obtained by two-sided testing that were less than 0.05 were considered significant. Multiple testing within subgroups was avoided and all statistical tests were performed for the complete data set. The statistical software, R v2.15.2,

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was used, and it included the ‘survival’ package (with survival analyses performed using log rank tests), the ‘stats’ package (including Fisher tests, Student t-tests, Mann-Whitney tests, and k-means analyses), and the ‘oncomodel’ package to calculate maximum likelihood tree models [34].

Analysis of net chromosomal changes: The data set was divided into two groups depending on

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the presence of synchronous (RFS-BM ≤ 1 months) versus metachronous (RFS-BM > 6 months) BM. There were no BM events that occurred between two and six months following a diagnosis. Differences in the overall numbers of net chromosomal changes, gains, and losses between the two groups were analyzed using t-tests and Mann-Whitney tests. For losses or

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performed.

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gains at each individual chromosome arm, Fisher tests for 2 × 2 contingency tables were

Oncogenetic trees: Briefly, oncogenetic trees of cytogenetic data represent probabilistic reconstructions that relate the occurrence of individual copy number changes to tumor progression [34]. The observed net changes are each represented as a terminal leaf of the tree and are placed according to their conditional probability of occurrence. The inner nodes represent hidden events, and the leaves closer to the root represent more frequent events. The degree of overlap of the leaves in a path to the root is a measure of positive correlation within the data set. When complete follow-up data was available, oncogenetic models were generated for the BM cases, as well as for the synchronous and metachronous BM subgroups. Only chromosomal imbalances that occurred with a frequency > 20% in each respective subgroup were considered. In addition, individual tree models were computed for cases that were included in each of the three k-means clusters.

ACCEPTED MANUSCRIPT K-means clustering: A binary matrix containing the most frequent chromosomal imbalances (e.g., those with a frequency > 20%) in the columns and the cases in rows was subjected to kmeans clustering. The number of clusters to be formed was set at k = 3, the maximum number of iterations was set to 100, and the number of restarts was set to 1,000 in order to make the results independent of the random starting points used. K-means assigns every case to one cluster. Survival curve difference tests were performed by testing each cluster group against a

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combined group consisting of the two other clusters.

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Results

Clinicopathological data and outcome Table 1 summarizes the clinicopathological data for the 148 cases of BM with ccRCC that were analyzed. The male to female ratio was 2.7:1 and the mean age [± standard deviation (SD)] at the time of surgery for BM was 60.1 ± 10.5 y (range 25–95 y). In 25% of the cases examined, BM events were diagnosed before, or within one month after, a nephrectomy of the primary

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ccRCC. No BM events were detected within 2–6 months of a diagnosis. Of the BM events analyzed, 75% were metachronous and were diagnosed at least six months following the first diagnosis of the primary ccRCC (range: 7–294 months). RFS-BM follow-up data were available for 68 patients, while OS data were available for 44 patients. The median RFS-BM for these

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cases was 31 months, and the 5-year RFS-BM was 35% (Fig. 1A). The median OS was 102.0 months and the 5-year OS was 63% (Fig. 1B). While there were no gender-related differences in RFS-BM, females tended to have a longer OS period than males (median: 112 vs. 86

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months, respectively; plogrank = 0.67). Moreover, 81% of BM events were supratentorially localized and did not influence RFS-BM or OS. Multiple BM events were detected in 11% of patients, and these patients experienced a significantly shorter OS period (p = 0.025). In addition, synchronous occurrence of BM significantly correlated with a shorter OS period compared with metachronous cases (p < 4 × 10-6).

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CGH

Supplemental Table 1 lists the CGH karyotypes for this series (n = 148). The mean chromosomal copy number change was 9.5 +/- 6.28 (median, 9.0). Genomic losses (mean, 5.56 +/- 3.86; median, 5.0) were observed more often than gains (mean, 3.9 +/- 3.44; median, 3.0).

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In addition, chromosomal imbalances occurred in at least 20% of all of the cases, and these involved gains at +5q (39%), +7q (34%), +7p (31%), +3q (30%), +8q (30%), +12q (28%), +5p

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(26%), and +12p (26%), and losses at -3p (81%), -14q (52%), -9q (50%), -9p (47%), -8p (41%), 18q (30%), -4q (22%), and -18p (22%). There were no statistically significant differences in the overall number of net chromosomal changes, including gains and losses, between the synchronous and metachronous BM events. In contrast, losses at 9p (p = 0.048) and 9q (p = 0.048) were significantly more frequent in metachronous brain metastases than in synchronous brain metastases of ccRCC.

K-means clustering and oncogenetic tree models Initially, unbiased k-means clustering was used to identify potential patterns of chromosomal imbalance. For this purpose, a data matrix of chromosomal imbalances that were detected in more than 20% of all of the 148 BM events were grouped into three clusters (Fig. 2, B-D). Subsequently, separate oncogenetic tree models were computed for each cluster (Fig. 2, E-G).

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These clusters had the following characteristics that were illustrated by the respective oncogenetic tree models representing each cluster. Cluster 1 was composed of 55 cases and these cases exhibited comparatively low numbers of chromosomal imbalances. Apart from -3p (56%), all of the imbalances occurred at comparably low frequencies of approximately 20% or less. In contrast, Cluster 2 was composed of 51 cases that exhibited a strikingly high incidence of -9p (82%) and -9q (90%) events, similar to that of -3p (94%). Furthermore, apart from -14q occurring with an intermediate frequency of 65%, all other imbalances had frequencies of 35%

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and lower. Cluster 3 was composed of 42 cases that exhibited a remarkably high overall number of chromosomal imbalances. Specifically, -3p was the most prevalent (98%), and the remaining imbalances occurred with frequencies ranging from 74% to 36%. These included: -8p (76%), +5q (74%), -14q (79%), +8q (67%), -9q (67%), -18q (67%), +5p (62%), +7q (62%), -18p

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(62%), +7p (60%), +12p (60%), +12q (60%), -9p (57%), +3q (48%), -4q (40%), and +1q (36%). The ratio of synchronous to metachronous BM events was also disproportionately higher for Cluster 1 compared with Clusters 2 or 3. For example, 42.9% of the cases that composed

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Cluster 1 included synchronous metastasis events, compared with 16.7% for Cluster 2 and 17.7% for Cluster 3 (Fig. 5A). The accumulation of synchronous cases in Cluster 1 was also illustrated by the sharp decline in the survival curve for Cluster 1. Correspondingly, a statistically significantly shorter RFS-BM was observed for Cluster 1 compared with Clusters 2 and 3 (p = 0.02; Fig. 5E), and a significantly longer RFS-BM was associated with Cluster 2 vs. Clusters 1 and 3 (p = 0.02; Fig. 5F). This uneven distribution of synchronous cases supports the

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hypothesis that a classification system based on net cytogenetic changes can identify clinically

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relevant subgroups of BM in RCC.

ACCEPTED MANUSCRIPT Discussion

A distinct characteristic of ccRCC is the unpredictable development of BM events. Moreover, a BM can develop before ccRCC is diagnosed, or up to 20 years after a primary tumor diagnosis [5, 15-18, 35-38]. In the present study, 25% of the BM events analyzed occurred prior to, or within one month, of a diagnosis of primary ccRCC, while 38% of the BM events appeared five

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years or more after diagnosis of the primary tumor. The frequency of synchronous cases observed in the present study may have been biased by the fact that single synchronous metastasis rarely occur and that patients with multiple, small BM events are often treated with stereotactic irradiation [36]. The latter were not included in the present study. In addition, a

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delayed diagnosis of BM may have influenced the results of the metachronous tree, since up to 30% of BM events are asymptomatic at the time of their initial diagnosis [35] and cerebral

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imaging is not routinely performed for asymptomatic RCC patients [39, 40].

Little is known about which chromosomal changes are critical for the development of distant metastasis with ccRCC. Genome-wide analyses indicate that losses at chromosomal arms 3p and 14q, as well as gains of 5q and 7, are indispensable for the development of ccRCC [19, 24, 27, 31, 41, 42]. Moreover, losses at 1p, 4p, 4q, 8p, 9p, 9q, 13q, 14q, and 18q have been found to correlate with higher malignancy grades and tumor stages in ccRCC [26, 27, 30-32, 42, 43].

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Although loss of 3p is the most common aberration associated with ccRCC both in primary lesions and metastases, 3p without detectable chromosomal changes, as well as losses at 4, 9p and 14q have been shown to serve as independent prognostic factors for RFS and OS in

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ccRCC [19, 31, 32, 44-46].

To our knowledge, the present study represents the largest series of ccRCC BM cases

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analyzed for chromosomal imbalances using CGH. There are only two other CGH reports for RCC brain metastases, and these include a total of five RCC BM cases [29, 33]. Petersen et al. reported four cases of BM events, and these included losses at 3p and 14q, as well as gains at 5q and 7q [33]. Bissig et al. analyzed one BM case, and losses at 8p and gains at 2p, 5p, 5q, 15q, and 17q were detected [29]. Consistent with previous reports of primary ccRCC, the most prevalent chromosomal imbalance in the present series of 148 ccRCC BM cases were losses at 3p (81%), followed by losses at 14q, 9, and 8p, as well as gains at 5q in over 50% of cases. In addition, losses at 18q and gains at 3q, 7, and 8q were detected in ~30% of the ccRCC BM events analyzed. Similar findings were reported for 31 bone metastases of ccRCC, and these included losses at 3p, followed by losses at 8p, 9, and 14q, together with gains at 5 and 8q [47]. In contrast, losses at 6 and gains at 17, which have been recurrently observed in ccRCC bone metastases [47], were not identified in the present series. Certain chromosomal aberrations

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have been reported to occur more frequently in lymph node and lung metastases compared with the corresponding primary ccRCC lesions, and these included losses at 8p and 9p [28, 29, 43]. These studies [27,28,42] also reported gains at 17q and Xq as prominent markers for lung and lymph node metastases of ccRCC, and these were not observed in the present BM series.

A key consideration in the present study was whether patterns of chromosomal imbalance for BM events of ccRCC could provide prognostic significance. Metastatic cancer specimens are

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generally heterogeneous entities that exhibit considerable intra-patient and inter-patient variations [48, 49]. Accordingly, probabilistic oncogenetic models were applied, thereby allowing the data to be fitted to a model of evolution that permitted multiple cytogenetic pathways to occur simultaneously in a given data set without assuming a unique order of events [34]. In

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general, oncogenetic trees provide a model for continuous cytogenetic evolution, despite the fact that a given tumor can only be observed once, typically at the time of surgery. By applying oncogenetic tree models to primary ccRCC, distinct pathways of cytogenetic evolution have

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been proposed. These include losses at 8p, 9p, 14q, and 18q, and these have also been associated with a less favorable outcome [30, 31, 34, 43].

Oncogenetic models consider each chromosomal aberration as an individual time point in evolution, and during construction of a tree, the most frequent aberrations are placed closest to the root of the tree. As the present data set represents a highly selected set of BM events of

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advanced stage ccRCC, it is important to note that cytogenetic alterations that have been described to occur late in the development of ccRCC, such as losses at chromosome 9, were placed close to the tree root. Overall, the oncogenetic tree analysis performed indicates that cases with BM showing early or late recurrence, respectively, may undergo different evolutions

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of chromosomal alterations. Based on these data, it is hypothesized that cases involving synchronous BM events may represent a subgroup of tumors that are more aggressive

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clinically, and are characterized by gains at chromosomes 5, 7, and 12. Moreover, these gains may initiate the development of metastasis. Conversely, late BM events of ccRCC may be explained by a model of evolution where tumor cells develop a chromosomal profile that is characteristic of advanced stage ccRCC, and this may include losses at chromosome 9 concomitant with losses at 14q and 8p.

A k-means analysis of chromosomal aberrations using three clusters provided more precise insight into the prognostic significance of chromosomal patterns in BM of ccRCC. For example, the cases in Cluster 1 generally exhibited a low number of chromosomal changes, a lower frequency of -3p, and the highest number of synchronously diagnosed BM cases. It cannot be excluded that tumors without deletion of 3p may harbor loss of heterozygosity (LOH) of 3p, as CGH cannot evaluate LOH status. Moreover, chromosome 3p has been shown to harbor factors

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frequently mutated or to undergo biallelic losses in ccRCC. In particular, the VHL tumor suppressor protein (pVHL) [50-52], as well as regulators of transcription such as the H3K36 methyltransferase, SET2D [48, 53], and a member of the ATP-dependent chromatin remodeller SWI/SNF, PBRM1 [54], have been affected. Functional inactivation of pVHL is commonly inheredited and has been detected in a majority of sporadic ccRCC cases. pVHL is part of the E3 ubiquitin ligase complex which targets the transcription factor, hypoxia inducible factor (HIF1A), for degradation [55]. Furthermore, in the absence of pVHL, HIF1A induces a

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transcription state that is characterized by synthesis of glycogen [56] and production of vascular endothelial growth factor (VEGF) [57], similar to that observed during hypoxia. In addition, in renal tubules and early lesions of VHL patients, HIF activation can precede clear cell morphology, thereby suggesting that tumor development requires the interplay of multiple HIF-

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activating events [58]. HIF-dependent effects can be pharmacologically counteracted by inhibiting VEGF receptor tyrosine kinase [59], as well as by inhibiting mammalian target of rapamycin (mTOR) kinase [60]. The latter is involved in signaling pathways related to cell

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growth/survival and also reduces translation of HIF1A [61]. However, neither anti-VEGF nor anti-mTOR-based therapies have been able to cure late stage ccRCC due to the development of drug resistance [62].

Cluster 2 exhibited a high occurrence of -9p/-9q (94–98%) and was mainly associated with metachronous metastases. Previously, loss of 9p has been detected more often in metastases

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than in primary tumors. Correspondingly, this loss represents a copy number change in primary tumors that is associated with a risk of metastasis [29, 32, 43, 63-65]. The results of the present study further indicate that loss of 9p may also characterize a comparatively low level of ccRCC metastasis aggressiveness, since -9p was mainly observed in metachronous BM cases. The

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HIF1A-target, carbonic anhydrase IX (CA9), is a gene located on 9p13, and low CA9 expression levels have been found to predict a shorter survival period for patients with metastatic ccRCC

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[66-68]. Another locus of interest is INK4a/ARF on 9p21. This region comprises several important tumor suppressors such as CDKN2A. Moreover, proteins encoded by these tumor suppressor genes control the functions of retinoblastoma tumor-suppressor, RB1, and p53 [69]. Furthermore, these genes not only predict prognosis in RCC, but also predict the prognosis of other solid tumors [70, 71]. Losses at 9q may also involve tumor suppressors located at 9q22, including PTCH1 [72] and 9q34, which encompass the TSC1 and TSC2 genes downstream of AMP-activated protein kinase, and also negatively regulate mTOR in response to cellular energy deficit [73].

Cluster 3 mostly included metachronous cases, and in approximately two thirds of these cases, losses at -9p/-9q were detected. However, in contrast with Cluster 2, Cluster 3 was characterized by a generally higher frequency of copy number changes, including common

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chromosomal changes such as -14, +5, +7, and +12. Consistent with the apparently advanced stages of cytogenetic evolution that were observed for Cluster 3, patients of this cluster also exhibited a shorter maximum RFS-BM of about eleven years, compared to a maximum RFS-BM of 25 years

for patients of Cluster 2. These results suggest that the overall number of

aberrations might represent a risk factor independent of the losses at chromosome 9. Loss of chromosome 14 has also been associated with a poor outcome in ccRCC [42, 44-46]. Reduced HIF1A activity has been detected in 14q-deleted kidney cancers, and all of the somatic HIF1A

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mutations identified in kidney cancers tested to date include loss of function [74]. Accordingly, deregulation of HIF target genes, such as VEGF, represents an abnormality characteristic of pVHL-defective neoplasms.

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One of the challenges of the present study was obtaining primary ccRCC tumor specimens, particularly those that were resected more than ten years before the development of brain metastases. Since the primary tumors were not analyzed, the present data do not distinguish

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between aberrations that were present in the primary lesion versus those that developed at distant metastatic sites, particularly those in the brain. Consequently, the chromosomal clusters described herein potentially characterize metastatic cell populations at the point of separation from the primary lesion. Undirected or scattered spread of tumor cells may represent one of the early steps in tumor evolution in ccRCC. Moreover, this may occur in primary lesions that are more aggressive, and it may be accompanied by a cytogenetic profile similar to that of Cluster

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1. In contrast, data from Clusters 2 and 3 suggest that tumor cells may acquire losses at chromosome 9 in their late stages, thereby leading to the migration of tumor cells from the primary tumor and the development of distant metastases in the brain.

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In summary, for the present series, subgroups of BM in ccRCC were identified using unbiased clustering of copy number variations, and these differences correlated with differences in RFS.

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Thus, the present data may help select genes of interest for further study, particularly those that may serve to estimate the risk of occurrence for early or late cerebral metastatic disease in ccRCC.

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Acknowledgements

B.S. acknowledges financial support from the Danish Council for Independent Research,

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Medical Sciences.

ACCEPTED MANUSCRIPT Figure Legends

Figure 1: Kaplan-Meier estimates for all cases with available follow-up data. (A) Recurrence-free survival until BM (n = 68) and (B) overall survival (n = 44) of ccRCC are presented. Estimates are indicated with solid black lines and 95% confidence

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intervals are represented by dashed lines.

Figure 2: K-means analysis of individual chromosomal aberrations for k = 3 clusters. (A) Distribution of synchronous versus metachronous cases among the clusters. The

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synchronous cases were predominantly assigned to Cluster 1. (B-D) Oncogenetic tree models of cases assigned to Cluster 1 (B), Cluster 2 (C), and Cluster 3 (D). (EG) Recurrence-free survival analyses for Cluster 1 (E), Cluster 2 (F), and Cluster 3

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(G). The frequency of occurrence for each aberration are indicated in the Results.

ACCEPTED MANUSCRIPT References

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[74]

ACCEPTED MANUSCRIPT Table 1: Clinico-pathologic data in 148 patients with BM of ccRCC.

108/40 (2.7:1)

Mean age years (range)

60.1 (25-95)

Singular/multiple BM

89%/11%

Supratentorial/infratentorial

81%/19%

Synchronous /metachronus BM

25%/75%

RI PT

Male/Female (ratio)

31 (36.2 – 69.5)

SC

Median RFS-BM months (95% CI) (n=68)

102 (90.4 – 144.9)

Median OS months (95% CI) (n=44)

AC C

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ccRCC: clear cell renal cell carcinoma

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BM: brain metastasis; RFS-BM recurrence-free survival until BM; OS: overall survival;



AC C



EP



TE D

 

 

  



M AN U







SC

RI PT















ACCEPTED MANUSCRIPT







  







A

B

C

ACCEPTED MANUSCRIPT

-3p

early late

80

months

200

300

+5q +5p

+7q

+3q

-9p

0.8 0.6

0.6 0.4

p = 0.02

cluster 3 others

0.4

G

1.0

-3p

p = 0.66

0

0.2

EP

1.0

+12q -8p

-1p

0 100

+12p

+7p

cluster 2 others

0.2

0.6 0.4

p = 0.02

+8q

+5q -4q

AC C

0.8

cluster 1 others

recurrence-free survival

1.0

F

TE D

3

0.8

RI PT

M AN U

+8q +2q

cluster 3

-9q

SC

20

-8p

-9p -9q

+3q

recurrence-free survival

60 40

cluster 2

-14q

-10q

0

% cases in cluster

+3q -13q

-18p

-3p

-6q

2 cluster

-18q

-18q

+5p -18q

1

-4q -14q

-3q

+12q

0.2

recurrence-free survival

+7p -10q

+5q

cluster 1

+7q

-8p

+12p

0

E

D

-14q

+7p +7q

100

months

200

300

100

200

months

300

ACCEPTED MANUSCRIPT

Appendix 1

Supplemental Table 1: Clinical data and chromosomal aberrations detected in 148 patients with BM of ccRCC.

OS

(months)

(months)

Gains

Losses

RI PT

Case Gender/Age RFS-BM

Cluster

1

m/69

n.a.

n.a.

+1q, +3q, +5, +7

-1p, -3p, -4, -14q, -18

3

2

m/57

184

221

+12

-3p, -6, -8pterq22, -

2

f/95

n.a.

n.a.

-

4

f/65

n.a.

n.a.

-

5

m/62

38

39

8

9

10

11

f/65

n.a.

24

m/51

m/46

m/62

n.a.

n.a.

n.a.

n.a.

1

-9, -14q

2

+1p11p31, +1q, +5,

-1p31pter, -3p, -

3

+6p, +7, +12

4q21qter, -8p, -10, -

+20p11p12, +21q21

18

+7q, +12, +13q, +19,

-1, -2q34qter, -3p, -

+20

4, -6, -7p, -8p, -9, -

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m/70

n.a.

n.a.

EP

7

m/62

AC C

6

-14q21q24

M AN U

3

SC

9, -14q

n.a.

n.a.

3

14q, -18, -21q

+1q, +5q14qter +7,

-1p, -3p, -4, -6, -8p, -

+8q, +11p, +12,

9, -10q, -11q, -14q, -

+17q +20

17p, -18

+3q, +5q15qter, +6p, -3p13pter, -8p, -9, -

3

2

+17q21qter

14q

+2q22q32,

-1p34p34, -3p, -9

2

-1p, -3p, -4, -5, -13q,

1

+7pter31, +13q n.a.

-

-15q, -18, -21q, -22q n.a.

+3q, +5q14qter, +7,

-3p, -8p, -9

3

+1p21p22,

-1p31pter, -

2

+5q23qter

3pterq13, -4q21q27,

+8q23q24, +12, +16 12

m/44

49

78

-5q12q14, -8, -9, -

ACCEPTED MANUSCRIPT 13

f/76

n.a.

n.a.

14q

+1q, +3q21qter, +5p, -3pterq13, +5q21qter

1

5q11q14, -13q, -14q, -17p, -17q21q21

14

m/49

0

0

-1p34pter, -

2

2q33qter, -3p, -

15

f/59

19

41

RI PT

8p12pter, -9 +1q, +3q, +21q

-1p32pter, -

2

3p12p24, -4q, -8p, -

SC

8q22q23, -9q, -14q, 16q, -18q, -22q

m/68

n.a.

n.a.

+1q25qter, +3q, +5q21qter

20

21

22

m/41

m/59

f/70

185

n.a.

n.a.

m/75

n.a.

212

n.a.

+1q32qter, +5p,

-1p34pter, -3p, -4, -

+5q31qter, +7

5q12q21-14q, -22q

+3p12qter,

-3p21pter, -13q, -

+8q21qter, +19q

14q, -18q

-

-3p13pter, -

TE D

19

m/61

n.a.

22q

n.a.

+1q21qter,

-1p32pter, 3p21pter, -14q

n.a.

+5q14qter,

-1p33pter, -

+12q13qter

3p14pter, -4, -9, -

n.a.

3

1

1

13q21qter

+7q21qter,

0

2

8pterq21, -10, -

EP

18

m/48

AC C

17

-3p14pter, 9q, 14q,

M AN U

16

1

+11p12pter 2

14q, -22q n.a.

+5, +7p, +12, +20

-3p14pter, -6, -8p, -

3

9, -14q, -18 23

m/53

n.a.

n.a.

+2q24q31,

-3pterq12, -9

3

-6q, -9p, -10, -

1

+7pterq22, +13q 24

f/64

22

136

-

12pterq12, -20p

25

m/71

121

26

m/58

7

ACCEPTED MANUSCRIPT n.a.

+7

-3p, -9p, -10, -14q

2

16

+3p13qter, +3q, +7,

-3p14p24

1

+12, +20, +21q m/57

n.a.

n.a.

-

-

1

28

m/88

n.a.

n.a.

+7q11q31, +12

-18q

2

29

m/66

n.a.

n.a.

+1q, +4p15pter, +8q, -3p, -6, -8p, +12pterq14,

9p13p21, -9q, -13q, -

+12q24qter, +16p,

14q, -20p

+20q m/57

140

151

+3q, +5q21qter, +7

-8p, -10q22qter, -

SC

30

RI PT

27

2

1

14q24qter

m/71

n.a.

n.a.

+3p13qter, +5, +7q,

-3p21pter, -9, -

+8, +11p12p14,

M AN U

31

10pterq22

+11p, +12p,

5pterq13, -6q, -8p, -

+17q22qter, +18p,

9q, -10p, -

+20q

11q22qter, -

3

+11q13q22, +12,

+13q14q31, +16p 32

m/66

n.a.

209

+1q, +2pterq32, +6p,

-1p, -3p14pter, -

3

m/54

n.a.

AC C

33

EP

TE D

+7, +9p, +10q24qter, 3q11q13, -4, -

n.a.

12q12qter, -13q, 14q, -15q

+1q, +2p, +3q, +5,

-1p, -3p, -4q, -6, -9, -

+7, +8q, +10, +12,

13q, -14q, -18q, -

+20p

21q, -22q

3

34

m/72

70

173

+5, +7, +12

-6q

1

35

m/46

0

8

+12q21qter,

-3p14pter, -

2

+15q11q11, +Xq

5q13q14, -9, -11q, 14q, -22q

36

f/55

27

n.a.

-

-3p, -14q

2

37

m/74

24

27

+7p, +11p, +20

-1p33pter, -

2

3pterq13, -4q, -9, -

ACCEPTED MANUSCRIPT

13q, -14q, -15q, 16q12qter, -18q

38

f/64

51

62

+1q, +2, +7

-3p, -4q23qter, -9, -

2

10, -16q 39

m/62

9

18

+8q, +10p, +20

-2q33qter, -3p, -

2

8p22pter, -9, -13q, -

40

m/55

197

202

RI PT

14q24qter, -18 +5q21qter

-3p14pter, -6, -9q, -

2

10p12pter, -

SC

10q23qter, -

11q22qter, -13q, 18q

m/65

97

139

-

M AN U

41

42

m/62

266

277

+5

43

f/62

n.a.

n.a.

-

m/70

n.a.

n.a.

n.a.

+3q, +5q32qter

TE D

45

m/57

n.a.

EP

44

+7

-3p21pter, -

1

10q21qter -3pterq21, -9, -18q

2

-3p14p24, -9, -

2

14q21q24 -3p14pter, -4, -6, -

1

8p, -10, -15q -3p, -5pterq15, -9, -

2

13q14qter

m/59

n.a.

n.a.

+3q, +7

-3p, -9, -14q

2

47

m/46

n.a.

n.a.

+3q, +5q15qter

-3p14pter, -9q, -

2

AC C

46

14q22qter

48

m/54

n.a.

n.a.

+7

-3p, -10q

1

49

m/62

n.a.

n.a.

+2q, +3q, +4q, +7

-3p21pter, -6p, -9q, -

2

10

50

m/72

n.a.

n.a.

-

51

m/57

n.a.

n.a.

+3p12qter (trend +4, -3p14pter, -8p, -9q, +5)

-

1 2

10q21qter, -13q, 15q

52

f/57

n.a.

n.a.

-

-

1

53

m/61

n.a.

54

f/77

n.a.

ACCEPTED MANUSCRIPT n.a.

-

-3p

1

n.a.

+4p, +5, +6pterq21,

-3p13p24, -

3

+6q24qter, +8q21,

4q24qter, -7p21pter,

+12, +20

-8p23q12, -9, 11q14qter, 11p13p14, -

RI PT

13q12q14, 13q22qter -

14q21qter, -18 m/58

n.a.

n.a.

+5q34qter, +8p12qter

-3p21pter, -

SC

55

2

4p15qter, -5pterq21, -9p13pter

57

m/82

m/70

n.a.

n.a.

n.a.

n.a.

+5, +7

M AN U

56

-1p, -3p21pter, -4q, -

3

8, -14q21qter, -18, 21q

+1q21q25,

-3pterq13, -

+2pterq21,

4q25qter, -6q, 8p

1

58

m/51

n.a.

TE D

+13q32qter

n.a.

+1q, +3q,

-3p, -6q, -8p, -18q

1

+5q31qter, +16q

-3p, -9pterq32, -14q

2

n.a.

+5, +20

-3p21pter, -18

1

79

-

-3p, -4p

1

27

-

-

1

+10p

-1p, -3p,

1

+5, +13q31q32

-1p, -3p, -4q15qter, -

3

+17q22qter

14

60

m/77

0

61

f/66

36

62

f/59

20

63

m/50

0

64

m/70

59

37

EP

m/66

AC C

59

73

8pterq21, -9, 14q21qter, -18

65

m/79

n.a.

n.a.

+5q14qter, +16p,

-3pterq22, -

+20

8p21pter, -9, -14q, -

2

21q 66

m/55

n.a.

n.a.

+2, +3q, +5, +8q,

-3p14pter, -

3

ACCEPTED MANUSCRIPT +12, +13q

8p21pter, -9q22qter, -10

67

m/66

n.a.

n.a.

+3p13qter, +5, +8,

-3p21pter, -9q, -

+Xq25qter

14q22qter, -22q

2

m/72

n.a.

n.a.

+8q

-

1

69

m/48

n.a.

n.a.

+5p, +5q23qter, +8q

-3p21p21, -8p, -9, -

3

RI PT

68

13q, -14q, -17p, -18 70

m/36

n.a.

n.a.

+7

-3p, -9p13pter

1

71

m/49

n.a.

n.a.

+10p11p12

-3p, -4q, -5q11q15, -

1

m/25

n.a.

n.a.

-

73

m/66

n.a.

n.a.

+1p13p31, +2q21q33,

-

1

-8p12pter, -

1

M AN U

72

SC

9p

10p12pter, -12, -15

+2q22q32,

-3p14pter, -

+6q21q24, +8q

10q22qter

+5q23qter, +7

-3p14pter, -8p, -9, -

+6q11q22, +8q13q23,

+10p11p11

75

m/62

m/59

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

+1q, +5p14qter, +7

EP

76

m/61

TE D

74

1

2

14q -1p32pter, -

3

3pterq13, -8p, -9p, 17p, -18p

m/56

n.a.

n.a.

-

-

1

78

m/67

n.a.

n.a.

-

-

1

79

f/66

n.a.

n.a.

-

-

1

80

m/40

n.a.

n.a.

+3q, +5, +7, +8q,

-3p21p21, -8p12pter

3

AC C

77

+12q14q21 81

f/64

n.a.

n.a.

-

-3p21pter, -8p

1

82

m/61

n.a.

n.a.

+1p31q21,

-3p14pter, -

3

+1q21qter, +3q, +5,

8p12pter, -9q, -

+7, +8q

10q21qter, -

ACCEPTED MANUSCRIPT

14q12qter, -18q

83

f/62

n.a.

n.a.

+5p13p14, +8q

-

1

84

f/61

n.a.

n.a.

+1q, +5q14q33

-1p, -3p14pter, -4, -

2

9, -14q22qter, -18 85

f/64

n.a.

n.a.

+5, +7, +8q12qter

, -3p21pter, -4q, -6,

3

RI PT

-8p, -9p13pter, -13q, -14q, -18

87

m/59

m/63

n.a.

n.a.

n.a.

n.a.

+5, +7, +12p12q21,

-3p14pter, -9, -14q, -

+13q14q32

18

-

-1p34pter, -

SC

86

3

2

3p14pter, -9

f/44

n.a.

n.a.

+3q

M AN U

88

-3p, -8p, -9p, -9q, -

2

14q

f/45

n.a.

n.a.

+3q, +8q

-3p, -8p, -9, -14q

2

90

f/63

0

n.a.

-

-

1

91

m/43

14

n.a.

-

-3p

1

92

f/72

0

n.a.

-

-3p21pter, -

1

f/52

20

94

f/51

0

96

AC C

95

m/64

m/53

0

34

10q22qter, 14q22qter

n.a.

-

-3, -9q, -14q, -18q

2

n.a.

+5, +8q21q23

-1p, -12p, -

1

EP

93

TE D

89

n.a.

15q14qter , 17, -18, 20 +1q, +3q, +5,

-3p14pter, -4, -6, -

+17q22q23, +20

14q13qter, -17p, -

3

18q n.a.

+1q, +2, +3q, +7

-1p, -3p14pter, -8, -

2

9p, -10, -14q, -15q 97

98

m/65

m/57

n.a.

0

n.a.

n.a.

+4q11q12, +12, +19,

-4q, -13q, -14q, -

+20

18q21qter

+2p11p16,

-3pterq13

+2q22qter, +7, +19,

1

1

ACCEPTED MANUSCRIPT +22q

99

m/52

23

n.a.

+1q,+7, +8q, +20

-3p21pter, -

3

6q11q21, -8p, -14q, 18 100

m/76

47

68

+2, +3p12qter, +5p,

-3p14pter, -6, -

+5q21qter, +8q, +12

8p12pter, -9, -10, -

3

101

m/64

161

162

RI PT

14q, -18 -

-3p14pter, -8p, -9, -

2

14q m/52

0

n.a.

+3q, +5q31qter, +8q, -3p, -8p, -13q, -14q, +10p, +12, +17q21qter, +20

105

106

f/74

32

24

26

294

m/61

13

+7, +8q, +12

M AN U

m/56

14

301

86

-3p21pter, -6, -

3

9p21pter, -8p, -13q, -14q -3p14pter, -

+8q21qter, +12,

6p21pter, -9q, -

+20p

14q22qter, -15q

+20q

-3p21pter, -8p, -

+5, +8q, +12

3

15q

+3q, +4p14, +5,

TE D

104

m/54

EP

103

SC

102

3

2

9q21qter, -14q, -18, -20p -3p, -8p, -9, -14q, -

3

18p

m/63

62

63

+3q, +8q, +12, +20q

-9, -22

2

108

m/51

0

29

+8q, +12, +20

-3p14pter, -22q

1

109

m/61

n.a.

n.a.

+5q32qter, +8q

-3p13pter, -9q, -

3

(trend +3p12qter)

14q21qter, -15q, -18

AC C

107

110

f/45

0

n.a.

-

-

1

111

m/73

0

n.a.

+5p14q22,

-2q34qter, -

1

+12q15q21,

3p21pter, -

+13q21qter

14q23qter, -16q

+12p11q21

-3p14p24, -

112

f/52

n.a.

n.a.

1

ACCEPTED MANUSCRIPT

8p12pter, 10q22qter

113

m/63

n.a.

n.a.

-

-

1

114

f/56

n.a.

n.a.

+5q21qter, +7, +16

-3p14pter, -8p, -9q, -

3

14q, -17p12pter, -

115

f/55

n.a.

n.a.

RI PT

18, -19 +8q12q23,

-3p21pter, -8p, -9,-

+13q13q31

10, -14q22qter, -

2

18q21qter m/na

n.a.

n.a.

+2q11q21+3q, +8q, +10pterq21, +11, +12, +17q22q24,

119

m/68

m/67

n.a.

n.a.

n.a.

n.a.

+7

TE D

118

m/53

n.a.

EP

117

M AN U

+20, +22q

n.a.

-2q22qter, -

3

SC

116

3p13pter, -4, 8p12pter, -9q21qter -14q, -16q

2q32qter, -3p21pter,

1

-4p15pter, -6, 8p12pter, 11q13qter, -13q, 14q22

+1q22qter, +7, +8q,

-1p, -3, -4, -6, -9, -

3

+10p, +12, +18p,

10q22q24, -14q, -

+20

18q

+3q21qter,

-3p14pter, -22q

1

+7q11q31, +12

m/70

n.a.

n.a.

+5q13qter

-3p, -4p, -9

2

121

f/63

60

n.a.

+3q, +5, +7

-3p, -6q14qter, -

3

122

123

AC C

120

m/58

m/67

1

93

8p21pter, -9, -13q, 18 17

111

+2, +5, +8q, +11,

-3p, -4, -6, -8p, -9, -

+14q

10q, -13q

-

-3p, -5pterq21, -9, -

2

2

15q 124

m/57

74

n.a.

+5p, +8q

-3p, -5q11q21, -

3

ACCEPTED MANUSCRIPT

6p22pter, -6q, -8p, 9, -13q, -14q, -17p, 18

m/65

28

n.a.

+5p, +12

-3p, -9, -10q, -13q

2

126

m/71

138

139

+1q11q41, +7,

-2q22qter, -

3

+8q23qter,

9q21qter, -13q, -

+12pterq24

14q, -18

127

m/56

n.a.

n.a.

RI PT

125

+5, +7, +8q, +12, +20 -3pterq13, -

3

8p21pter, -9, -

128

m/36

0

n.a.

+2pterq32, +3q, +7, +8q, +10p, +12

130

131

f/70

36

f/66

n.a.

44

m/68

112

7

12

+1q, +5q23qter

M AN U

129

SC

11q14qter, -14q

+1q, +5q31qter, +8q

+2, +3q, +7, +12,

-3p21pter, 8p

3

-3p21pter, -8p, -9, -

2

14q -1p, -3p, -8p, -

1

14q21qter -

1

-9

2

-3pterq13

1

132

m/45

211

TE D

+17q, +20q

219

+1q, +3q, +5q, +6q,

133

m/46

n.a.

+2q24qter,

EP

+7q, +8p, +12, +15q

n.a.

+16pterq21

m/36

n.a.

n.a.

+7, +10p,+15q

-8p

1

135

m/55

0

24

+2pterq21,

-2q23qter, -

2

136

AC C

134

f/74

n.a.

+3q24qter, +5p, +12, 3ppterq13, ++20q

4q22qter, -9, -14q, 20p

n.a.

+1q, +2pterq32, +3q, -1p34pter, +6, +7, +10p,

2q32qter, -3p, -

+17q22qter, ++5p,

5q13q21, -8p,- 9p, -

++8q

10q21qter, -11q, 13q, -14q, -18, -21q,

3

ACCEPTED MANUSCRIPT

-22q

137

m/na

n.a.

n.a.

+5q31qter

-3p, -14q

1

138

m/52

73

112

+2, +3q, +5q21qter,

-3p14pter, -4, -

3

+7, +8q, +11p, +12,

5pterq14, -8p, -9, -

+16, +20

14q, -15q, -

RI PT

17pterq12, -18 139

f/46

n.a.

n.a.

-

-

140

f/56

239

312

+3q21qter, +7

-3pterq13, -4, -5, -9,

1 2

-14q, -15q15q21, -

141

f/58

67

124

+3q26, +7

SC

17p

-3p, -4, -5, -

2

9pterq21, -14q

m/62

73

136

+7

143

f/62

87

93

-

f/57

107

n.a.

145

m/57

115

121

147

f/48

EP

m/57

102

AC C

146

n.a.

146

-3p

1

-2q33qter, -

2

3pterq21, -9p21pter, -14q11q22

-

-3p, -6q, -9, -15q

2

+3q, +12, ++8q

-3p, -4, -5p15q21, -6,

3

TE D

144

M AN U

142

+1q, +2p, +5, +8q,

-8p, -9p, -9q21qter, 11q14qte, -13q, 14q, -18, -21q -3p, -7, -8p, -9, -18

3

+1p32qter,

-1p35pter, -

3

+3q22qter,

3p14pter, -4q, -8p, -

+5q15qter, +7

9, -10q23qter, -14q,

+12, +21q21, ++5p11p14,

n.a.

-15q, -18, 21q11q21, -22q 148

m/70

127

n.a.

+3q, +5, +8q12qter,

-3p13pter, -8p, -14q,

+12

-18

3

ACCEPTED MANUSCRIPT

BM = brain metastasis; ccRCC = clear cell renal cell cancer; RFS-BM = recurrence-free survival until

AC C

EP

TE D

M AN U

SC

RI PT

brain metastasis; OS = overall survival; m = male; f =female; n.a.= not available