8p deletions in renal cell carcinoma are associated with unfavorable tumor features and poor overall survival

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Laboratory-Kidney cancer

8p deletions in renal cell carcinoma are associated with unfavorable tumor features and poor overall survival Till Eichenauer, M.D.a, David C. Bannenbergb, Martina Kluthb, Corinna Wittmer, M.D.b, Franziska B€ uscheck, M.D.b, Katharina M€ oller, M.D.b, David Dum, M.D.b, Christoph Fraune, M.D.b, Claudia Hube-Maggb, Christina M€ oller-Koopb, Roland Dahlem, M.D.a, Margit Fischa, Michael Rink, M.D.a, Silke Riechardt, M.D.a, Maria Christina Tsourlakis, M.D.b, Christian Bernreuther, M.D.b, Sarah Minner, M.D.b, Ronald Simonb,*, Guido Sauterb, Waldemar Wilczak, M.D.b, Till S. Clauditz, M.D.b a

Department of Urology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

b

Received 6 May 2019; received in revised form 2 July 2019; accepted 25 September 2019

Abstract Background and methods: 8p deletions are common in renal cell carcinoma. To study their prognostic impact and association with kidney cancer phenotype, a tissue microarray with 1,809 cancers was analyzed by fluorescence in situ hybridization for 8p21 copy numbers. Results: One thousand four hundred and seventy four interpretable tumors showed substantial differences between renal cancer subtypes. That 8p deletion was only seen in 1 (0.5%) of 216 papillary carcinomas underscores the biologic uniqueness of papillary kidney cancer, which is also defined by a highly distinct morphology. 8p deletions were found in 13.2% of 976 clear cell carcinomas, 7.8% of 77 chromophobe carcinomas, 0.8% of 119 oncocytomas, but also in several rare tumor entities including 1 of 4 collecting duct cancers, 1 of 3 multilocular cystic clear cell renal cell neoplasm of low malignancy, 2 of 10 Xp11.2 translocation cancers, 3 of 18 not otherwise specified carcinomas, and 1 analyzed medullary carcinoma. In clear cell carcinomas, 8p deletions were significantly associated with higher International Society of Urologic Pathologists (ISUP) grading (P = 0.0014), Fuhrman (P = 0.0003) and Thoenes grade (P = 0.0033), advanced tumor stage (P = 0.0002), large tumor diameter (P = 0.0019), distant metastases (P = 0.0183), overall survival (P = 0.0394), and recurrence free survival (P < 0.0001). In multivariate analysis, the prognostic role of 8p deletions was not independent of established clinic-pathological parameters. In conclusion, 8p deletions are strongly linked to tumor aggressiveness in clear cell kidney cancer. Conclusions: Because 8p deletions are easy to measure by fluorescence in situ hybridization, 8p deletion assessment, most likely in combination with other parameters, may have a role in future prognosis assessment in clear cell kidney cancer. Ó 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Keywords: Renal cell cancer; 8p21 deletion; Fluorescence in situ hybridization; Tissue microarray; Prognosis

1. Introduction In 2012, approximately 84,400 new cases of renal cell carcinoma (RCC) have been diagnosed in the European Union and cancer related deaths were estimated at 34,700 [1]. Over the past decades, kidney cancer incidence has continuously risen worldwide [2] particularly in developed *Corresponding author. Tel.: +49-40-7410-57214; fax: +49-40-7410-55997. E-mail address: [email protected] (R. Simon).

countries [3]. The increasing incidence might be driven by rising influence of established risk factors including increasing age, smoking, obesity, and hypertension, but also the better availability of imaging modalities like ultra sound, computer tomography, and magnetic resonance imaging [2]. Whereas the incidence is rising, mortality declines particularly in more developed countries [3]. Localized RCCs are generally treated either by radical or partial

https://doi.org/10.1016/j.urolonc.2019.09.024 1078-1439/Ó 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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nephrectomy. Because of its resistance to radiation and classical chemotherapy, nephrectomy and even metastasectomy are performed in some cases of metastatic disease. In patients with unresectable metastases, targeted therapies are used for the treatment of metastatic disease [4]. New therapeutic agents have indeed shown to improve the prognosis of patients with metastatic RCC [5,6]. Whether these new drugs might also be useful to reduce disease recurrence and improve survival in an adjuvant setting is currently being analyzed in several studies (Keynote-564, iMmotion010, Checkmate-914). So far, sunitinib is the only drug that has been approved by the FDA for adjuvant therapy of high risk RCC patients after nephrectomy [7]. Given these new therapeutic options, the accurate identification of highrisk patients becomes increasingly important. Kidney cancer aggressiveness is currently estimated based on pT, pN, and histological grade [8]. These parameters suffer from limitations such as interobserver variability of grading and the arbitrary definition of pT categories. If the individual risk assessment decides on whether or not adjuvant systemic therapy is applied in kidney cancer patients, better and more reproducible criteria will be needed to identify patients in need for such treatments. As the molecular mechanisms is involved in kidney cancer increasingly known, it can be hoped, that the analysis of appropriate molecular parameters will eventually result in better prognostic parameters [8]. 8p deletions are of interest in this context. They belong to the most common deletions in RCC [9]. In 1 study on 105 cancers, an association of 8p deletions and unfavorable kidney cancer phenotype was suggested [10]. To gain more information about a possible diagnostic and therapeutic utility of 8p deletions, we evaluated 1,809 RCCs with clinical follow-up data in a tissue microarray (TMA) format in this study. 2. Materials and methods 2.1. Patients Our set of kidney tumor TMA contained one 0.6 mm tissue core each from 1,809 kidney tumors. All samples were from patients who underwent surgery between 1994 and 2016 and whose tumors were histopathologically evaluated at the Institute of Pathology of the University Medical Center Hamburg-Eppendorf, Germany. All tumors had been reviewed according to the criteria described in the 2016 WHO classification by 2 pathologists with a special focus on genitourinary pathology (F.B., C.F.) and ISUP grading was performed for each tumor. The TMA is composed of 4 blocks, one of which had been earlier constructed [11]. The TMA manufacturing process was described in detail [12]. From each tumor, 1 tissue core measuring 0.6 mm in diameter was taken from a cancer containing tissue block. Clinical and pathological parameters of the arrayed cancers are summarized in Table 1. The mean follow-up time was 48 months. The usage of archived diagnostic left-over

Table 1 Pathological and clinical data of the arrayed renal cell tumors. Study cohort on TMA (n = 1809) Follow-up Available (n) 1174 Mean (mos) 48 Median (mo) 61.8 Age (y) <50 263 50−70 951 70−90 595 Histology Clear cell RCC 1167 Papillary RCC 270 Chromophobe RCC 101 Oncocytoma 149 Union internationale contre le cancer (UICC) stage I 733 II 131 III 175 IV 158 pT category pT1 998 pT2 223 pT3−4 408 International Society of Urologic Pathologists (ISUP) grade 1 398 2 537 3 469 4 100 Fuhrman grade 1 72 2 851 3 480 4 110 Thoenes grade 1 497 2 839 3 177 pN category pN0 232 PN+ 49 pM category pM0 220 pM+ 148 RCC = renal cell carcinoma; TMA = tissue microarray. Numbers do not always add up to 1809 in the different categories because of missing data.

tissues for TMA manufacturing analysis for research purposes as well as patient data analysis has been approved by local laws (HmbKHG, x12,1) and by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki Declaration. 2.2. Fluorescence in situ hybridization (FISH) The FISH procedure was exactly as previously described [13]. More details are provided in the supplementary

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results. For FISH analysis interpretation, the predominant FISH signal numbers were recorded for each tissue spot. Only tumor cells were analyzed for 8p deletion. Normal cells were used for internal control of the hybridization success. Tissue spots lacking 8p signals in all (tumor and normal) cells or tissue spots lacking normal cells were excluded from the analysis. Heterozygous 8p deletion was defined as presence of fewer 8p probe signals than centromere 8 probe signals in ≥60% tumor nuclei. Complete absence of 8p signals in the tumor cells, but presence of centromere 8 signals in tumor cells and of centromere 8 and 8p signals in adjacent normal cells, was required to define a homozygous deletion. These thresholds were based on a previous study comparing Phosphatase and Tensin homolog (PTEN) deletion data obtained by FISH and single nucleotide polymorphism (SNP) array analysis [14]. 2.3. Statistics Statistical calculations were performed with JMP 12 software (SAS Institute Inc, NC, USA). Contingency tables and the Chi-square test were performed to search for associations between 8p deletions and tumor phenotype. Survival curves were calculated according to Kaplan−Meier. The log-rank test was applied to detect significant survival differences between groups.

Fig. 1. Representative images of FISH analysis. (a) normal 8p21 copy numbers (absence of 8p deletion) as indicated by 2 orange 8p21 signals and 2 green centromere 8 signals and (b) presence of heterozygous deletion as indicate by the lack of 1 orange 8p signal. FISH = fluorescence in situ hybridization. (Color version of figure is available online.)

3. Results 3.1. Technical aspects One thousand four hundred and seventy four of 1,809 tumors were analyzable (81%). Reasons for noninformative TMA-spots were a complete lack of tissue, absence of cancerous tissue in TMA spots or insufficient quality of hybridization.

3.2. 8p deletions and renal cell tumor subtypes 8p deletions were found in 145 of 1,474 (9.8%) analyzable tumors. Deletions were always heterozygous. Representative images of deleted and undeleted cancers are shown in Fig. 1. The frequency of deletions varied between kidney tumor subtypes. Of 976 analyzable clear cell carcinomas, the most common subtype of kidney tumors, 129 (13.2%) featured 8p deletions. In contrast, 8p deletions were found in only 1 of the 216 papillary carcinomas (0.5%), as well as in 1 of the 119 oncocytomas (0.8%). In chromophobe carcinomas, 6 of 77 (7.8%) tumors had 8p deletions. Deletion were not seen in 26 clear cell tubulopapillary RCCs. 8p deletions were occasionally also seen in some less common tumor subtypes, and there were other entities in which we did not find any 8p deletion (Table 2).

Table 2 Prevalence of the presence of 8p21 deletions in different histological subtypes of renal cell tumors. Renal cell tumor type

Analyzable (n)

8p deletion (%)

Clear cell renal cell carcinoma Papillary renal cell carcinoma Oncocytoma Chromophobe renal cell carcinoma Clear cell (tubulo) papillary renal cell carcinoma Carcinoma no otherwise specified (NOS) Nephroblastoma Xp11.2 translocation renal cell carcinoma Collecting duct carcinoma Multilocular cystic clear cell renal cell neoplasm of low malignancy Metanephric adenoma Tubulocystic renal cell carcinoma Acquired cystic disease-associated renal cell carcinoma Cystic nephroma / mixed epithelial stroma tumor Medullary carcinoma Mucinous tubular and spindle cell carcinoma Reninoma

976 216 119 77 26

13.2 0.5 0.8 7.8 0

18

16.7

15 10

0 20.0

4 3

25.0 33.3

2 2 1

0 0 0

1

0

1 1

100 0

1

0

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tumor grades irrespective of whether ISUP (P = 0.0014), Fuhrman (P = 0.0003), or Thoenes (P = 0.0033) grading were used (Table 3). The lymph node status was not significantly correlated to 8p deletions (Table 3). In addition, 8p deletions were associated with larger tumor size (P = 0.0019, Table 4) and higher patient age at the time of diagnosis (P = 0.0346) in clear cell RCC.

Table 3 Associations between the presence 8p21 deletions and pathological parameters of clear cell renal cell carcinomas. Clear cell carcinomas Analyzable (n) All 976 UICC I 430 II 68 III 122 IV 107 ISUP 1 260 2 323 3 316 4 68 Fuhrman 1 50 2 528 3 320 4 77 Thoenes 1 330 2 518 3 127 Tunor stage pT1 582 pT2 112 pT3-4 275 Lymph node metastasis pN0 140 pN1 9 pN2 21 Distant metastasis pM0 127 pM1 106

8p deletion

P value

13.2 9.1 10.3 22.1 23.4

<0.0001

10.1 10.8 14.2 29.4

0.0014

12.0 9.7 16.0 27.3

0.0003

10.3 12.7 22.8

0.0033

9.8 14.3 20.4

0.0002

17.9 44.4 19.1

0.2091

12.6 24.5

0.0183

3.4. 8p deletions and patient outcome 8p deletions were linked with unfavorable disease course in clear cell carcinoma patients (Fig. 2). Outcome differences resulted in statistically significant differences for overall survival (P = 0.0394) and progression free survival (P < 0.0001) but narrowly failed to reach statistical significance for tumor specific survival (0.0654). In a multivariate analysis including pT, pN, M status, and ISUP grade, 8p deletions failed to achieve independent prognostic relevance for all endpoints (Table 5). 4. Discussion The 8p deletion frequency was 10% in this study. This is lower than in 19 earlier published studies describing 8p deletions in 15% to 77% of analyzed kidney cancers using either conventional cytogenetics [15], comparative genomic hybridization [9, 16-23], SNP arrays [24], loss of heterogeneity (LOH) analyses [10,25-28] or next generation sequencing [29]. We believe that these differences are largely caused by methodological differences as our present study is the first to apply FISH for 8p deletion analysis. FISH represents the gold standard for deletion detection as it enables exact gene copy number assessment on a cell by cell basis including a clear-cut distinction of losses and gains of chromosomal material. Methods analyzing extracted DNA like comparative genomic hybridization, SNP arrays or LOH may overestimate the true frequency of deletion because chromosomal imbalances due to trisomy or polysomy cannot be reliably distinguished from deletions. Indirect validation for our data comes from an earlier study using the same FISH procedure for 8p analysis

3.3. 8p deletions and tumor phenotype As clear cell RCC was the only subtype with more than 6 deleted cases, all subsequent statistical analyses were restricted to clear cell RCCs. In this subtype, 8p deletions were significantly linked to advanced tumor stage (P = 0.0002), distant metastases (P = 0.0183), and higher

Table 4 Role of the presence of 8p deletions for tumor size and patient age in clear cell renal cell carcinomas. 8p status

All tumors Clear cell RCC Papillary RCC Chromophobe RCC

RCC = renal cell carcinoma.

Normal Deletion Normal Deletion Normal Deletion Normal Deletion

n

1305 143 835 127 210 1 70 6

Tumor size (cm)

n

Mean § sd

P value

5.1 § 0.1 6.3 § 0.3 5.2 § 0.1 6.1 § 0.3 5.1 § 0.3 11 § 3.6 4.9 § 0.4 7.5 § 1.2

<0.0001 0.0019 0.0815 0.0404

1330 145 847 129 215 1 71 6

Patient age (y) Mean § sd

P value

62.2 § 0.4 65.5 § 1.1 63.5 § 0.4 65.7 § 1.0 61.0 § 1.0 80.0 § 14 58.8 § 1.5 67.0 § 5.0

0.0056 0.0346 0.1761 0.122

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a

8p normal (n=572) 8p deletion (n=88) p=0.0394

b

8p normal (n=572) 8p deletion (n=88) p=0.0645

c

8p normal (n=532) 8p deletion (n=84)

p<0.0001

Fig. 2. Association between presence or absence of 8p deletion and (a) overall survival (OS), (b) tumor-specific survival (TSS) and (c) progression-free survival (PFS) in clear cell renal cell carcinomas. Normal = absence of 8p deletion.

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in prostate cancer [13]. Our 8p deletion frequency obtained by FISH was about 3 times lower in kidney cancer (10%) than in prostate cancer (36%). The same ratio (threefold) was seen for 8p deletions in kidney cancer (21%) and prostate cancer (65%) in The Cancer Genome Atlas data available from the cBio Cancer Genomics Portal [29], the largest database containing data obtained by standardized methods on extracted tumor DNA. This supports the notion of these differences between FISH data and extracted DNA data reflecting an inherent methodological issue. Chromosome 8p deletion belongs to the most common deletions in cancer (reviewed in [30]). The vast majority of 8p deletions involve the entire short arm of the chromosome. For the purpose of this study, we had selected an NKX3.1 [31] FISH probe, because the gene is a putative tumor suppressor gene located in the center of 8p deletions. However, extensive studies searching for 8p tumor suppressor genes have identified a large variety of further 8p genes with tumor suppressive properties, including for example PPP2CB, PPP3CC [32], DLC1 [33], CSMD1 [34], RHOBTB2, TNFRSF10C and TNFRSF10D [32], and MSR1 [35]. Extensive sequencing efforts have failed to identify any recurrent gene mutations on 8p in kidney cancer and other tumors, indicating, that 8p deletion is usually not accompanied by a mutation of any 8p gene [36-39]. It thus appears possible, that reduced expression of multiple 8p genes could jointly exert a cancer promoting function. In murine models of hepatocellular carcinomas, also frequently affected by long 8p deletions, partial inactivation of multiple 8p genes was shown to cooperatively induce cancer development [40]. A similar model of compound haploinsufficiency might also apply for 8p-deleted RCCs. The successful analysis of 216 papillary kidney carcinomas demonstrates that 8p deletions are virtually absent in this tumor type. This is highly uncommon as 8p deletions occur at considerable frequency in virtually every cancer type [29]. It might be speculated that so far unknown elements of the specific cellular microenvironment of papillary RCCs prevent the development of 8p deletions or the survival of affected cells. This observation provides another argument for unique biologic properties of papillary kidney cancers which are also characterized by a unique and highly characteristic morphology. It is of note that the rate of only 13.2% 8p deleted clear cell carcinomas and the occurrence of 8p deletions in several rare kidney cancer subtypes preclude the use of 8p deletions as a tool for kidney cancer subtype diagnosis. The results of our study identify 8p deletions as a potentially useful prognostic feature in clear cell kidney cancer. These data are in line with an earlier report describing an association of 8p deletion with high grade and stage in 105 kidney cancers analyzed by LOH measurement [25]. Our data also fit with data from several other tumor entities. In a study on more than 7,000 prostate cancers, we identified 8p deletions as an independent predictor of biochemical

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Table 5 Multivariate analysis in clear cell renal cell cancers using the endpoints overall survival (OS), tumor specific survival (TSS), and progression-free survival (PFS)

pT pN pM ISUP

8p

3−4 vs. 2 2 vs. 1 N2 vs. N1 N1 vs. N0 M1 vs. M0 4 vs. 3 3 vs. 2 2 vs. 1 del. vs. norm.

OS

P

TSS

P

PFS

P

2.7 (1.0−8.1) 1.3 (0.4−3.8) 1.5 (0.5−5.3) 0.9 (0.3−2.3) 1.8 (0.9−3.8) 1.0 (0.4−2.4) 1.8 (0.9−3.8) 2.3 (0.7−10) 0.8 (0.4−1.5)

0.0206

3.9 (1.0−26) 1.8 (0.2−15) 1.3 (0.4−6.1) 0.9 (0.2−2.8) 2.3 (0.9−6.3) 1.3 (0.5−3.5) 1.5 (0.6−4.3) 3.7 (0.6−71) 0.8 (0.3−1.9)

0.0159

2.2 (0.9−5.9) 1.7 (0.6−4.7) 1.0 (0.5−2.4) 1.8 (0.8−3.8) 2.6 (1-4−5.2) 1.4 (0.6−2.9) 1.3 (0.7−2.4) 0.4 (0.6−3.8) 1.0 (0.6−1.8)

0.0068

0.7345 0.129 0.0254

0.4827

recurrence [13]. In another study using the same FISH method, significant associations were found between 8p deletions and advanced tumor stage, higher grade, higher tumor cell proliferation, and shortened overall survival in breast cancer [41]. Other authors had found an association of 8p deletions with an unfavorable tumor phenotype and aggressive tumor behavior in many cancer types such as hepatocellular carcinoma [42-45], breast cancer [41,46,47], lung cancer [48], colorectal cancer [49], esophageal cancer [50], and urothelial carcinoma of the bladder [51]. We assume that 8p deletion assessment might gain practical importance in clear cell kidney cancer work-up, even though 8p deletions did not provide prognostic information that was independent of the established clinic-pathological parameters which were diagnosed by expert pathologists. This is because deletion analysis by FISH is an easy and reproducible method always resulting in a yes/no answer, which is different from histopathological diagnosis and immunohistochemical analyses. The precision of histological grading and staging largely depends on the pathologist’s experience and the quality and amount of tissue available for diagnosis, and immunostaining results depend on the used protocol and is subject to false-positive and false-negative results [52]. Patients with 8p deleted clear cell carcinomas were significantly older than those without deletions in this study. Few molecular cancer features are known to be age dependent. For example, TMPRSS2:ERG fusions occur more often in young prostate cancer patients [53]. Development of these fusions is dependent on active androgen signaling offering higher serum testosterone levels in young men as a possible explanation for this age predilection. Y chromosome losses have been described to preferentially occur in normal and neoplastic tissues of the elderly. It has been suggested that this chromosome with only 72 protein-coding genes is not needed in many mature tissues and losing it might reduce the DNA manufacturing burden in cell division and thus facilitate a clonal outgrowth of benign and malignant cells with a Y loss [54]. Partly because the target genes of 8p deletions are unknown, we cannot offer a biological explanation for an age dependency of these deletions. Since 8p deleted cancers were also larger than

0.9009 0.0839 0.1049

0.6183

0.0996 0.0032 0.2156

0.8691

undeleted tumors, it appears possible that age dependency reflects a later cancer detection of more advanced tumors in older patients. In summary, our data identify 8p deletions as a molecular feature with moderate prognostic value in clear cell kidney cancer. To date, there is no established molecular feature that has strong prognostic value in renal cell tumors on its own. We thus expect that a clinically relevant kidney cancer prognosis test must combine multiple parameters in the future. 8p deletion assessment may well be part of such a procedure. Acknowledgments We are grateful to Inge Brandt, S€unje Seemann, Melanie Witt, Maren Eisenberg, Sylvia Schn€oger, and Sascha Eghteshadi for excellent technical assistance. Supplementary materials Supplementary material associated with this article can be found in the online version at https://doi.org/10.1016/j. urolonc.2019.09.024. References [1] Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, Rosso S, Coebergh JW, Comber H, et al. Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer 2013;49: 1374–403. [2] Rossi SH, Klatte T, Usher-Smith J, Stewart GD. Epidemiology and screening for renal cancer. World J Urol 2018;36:1341–53. [3] Wong MCS, Goggins WB, Yip BHK, Fung FDH, Leung C, Fang Y, et al. Incidence and mortality of kidney cancer: temporal patterns and global trends in 39 countries. Sci Rep 2017;7:15698. [4] Ljungberg B, Albiges L, Abu-Ghanem Y, Bensalah K, Dabestani S, Montes SF, et al. European Association of Urology guidelines on renal cell carcinoma: the 2019 update. Eur Urol 2019;75:799–810. [5] Motzer RJ, Tannir NM, McDermott DF, Aren Frontera O, Melichar B, Choueiri TK, et al. Nivolumab plus ipilimumab versus sunitinib in advanced renal-cell carcinoma. N Engl J Med 2018;378:1277–90. [6] Choueiri TK, Halabi S, Sanford BL, Hahn O, Michaelson MD, Walsh MK, et al. Cabozantinib Versus sunitinib as initial targeted therapy for patients with metastatic renal cell carcinoma of poor or

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