CASE REVIEW. Oncocytic Renal Neoplasms. A Molecular Approach to a Common Diagnostic Challenge

CASE REVIEW Oncocytic Renal Neoplasms A Molecular Approach to a Common Diagnostic Challenge Federico A. Monzon, MD,* Steven S. Shen, MD, PhD,* Bonnie Kemp, MD, Karla Alvarez, BSc, and Alberto G. Ayala, MD* Abstract: Oncocytic features are a hallmark of renal oncocytoma (OC) but can be seen in other renal tumors such as clear cell renal cell carcinoma (ccrcc) with granular cells and eosinophilic variant of chromophobe RCC (chrcc). Up to 7% of renal neoplasms are ultimately diagnosed as unclassified RCC with oncocytic tumors accounting for a significant number of these. One of the common diagnostic challenges with renal oncocytic tumors is the differentiation between OC and eosinophilic variant of chrcc. The distinction between these 2 entities is critical due to the prognostic implications and patient management decisions. Immunohistochemistry is generally a useful tool for characterizing many renal tumors but is of limited utility in this situation, since staining patterns for these 2 neoplasms are quite similar. However, OC and chrcc have recurrent chromosomal abnormalities that are characteristic of each tumor type. The use of molecular chromosomal analyses as an ancillary tool for this diagnostic challenge has been reported. This case review focuses on the role of molecular analyses, such as virtual karyotyping with SNP arrays, in the diagnosis of unclassified oncocytic renal tumors. Key Words: renal cell carcinoma, unclassified, SNP array, virtual karyotype, chromosomal imbalance, oncocytoma, chromophobe, eosinophilic (Pathology Case Reviews 2010;15: 20 24) Renal cell carcinomas (RCCs) are morphologically and clinically heterogeneous. Distinction among RCC subtypes is generally straightforward during routine morphologic evaluation by light microscopy. 1 However, approximately 7% of renal tumors are ultimately diagnosed as unclassified RCC, due to the presence of nonspecific overlapping features that can be seen in all of the subtypes (such as oncocytic or sarcomatoid features), an unusual morphology that has not been described in known subtypes of RCC or the presence of combined morphologies in a given tumor. 1,2 One of the common diagnostic challenges in renal pathology is the differentiation between oncocytoma (OC) and eosinophilic variant of chromophobe renal cell carcinoma (chrcc). The distinction between chrcc, a malignant neoplasm with confirmed metastatic potential, and OC, a benign tumor, has significant medical and psychologic implications. Immunohistochemistry is of limited utility in this situation, since staining patterns for these 2 neoplasms are From the *Department of Pathology, The Methodist Hospital, Houston, TX; Department of Pathology, Weill-Cornell Medical College, New York, NY; Department of Pathology, The Methodist Hospital Research Institute, Houston, TX; and Department of Pathology, Memorial Hermann Memorial City Hospital, Houston, TX. Supported by (Departmental funding) the Department of Pathology at The Methodist Hospital and a Research Scholar Award from The Methodist Hospital Research Institute (TMHRI) (to F.A.M.). Reprints: Federico A. Monzon, MD, Department of Pathology, The Methodist Hospital and The Methodist Hospital Research Institute, 6565 Fannin St, MS205, Houston, TX 77030. E-mail: famonzon@tmhs.org. Copyright 2010 by Lippincott Williams & Wilkins ISSN: 1082-9784/10/1501-0020 DOI: 10.1097/PCR.0b013e3181d2d808 quite similar and frequently show overlap. 2,3 It has been shown that each renal cell tumor subtype shows characteristic chromosomal abnormalities, and therefore the use of molecular chromosomal analyses as a useful ancillary tool for this diagnostic challenge has been reported. 4 6 This case review focuses on the role of molecular analyses in the diagnosis of unclassified oncocytic renal tumors. CASE REPORT Clinical Features A 53-year-old woman presented with history of abdominal pain. A computed tomography (CT) scan of the abdomen revealed a 1.7-cm left renal mass possibly representing an enhancing neoplasm or a complex cyst. Magnetic resonance imaging (MRI) showed this lesion to be an enhancing mass in the posterior aspect of the left kidney which was hypointense on T1/T2 weighted images and was consistent with a renal neoplasm. The patient had no family history of renal cancer or any other pertinent significant clinical medical history. Based on the imaging diagnosis of a renal neoplasm, a left partial nephrectomy was performed. Pathology Grossly, the tumor measured 1.7 cm in greatest dimension and showed a homogeneous pale gray cut surface without evidence of hemorrhage or necrosis. The tumor was confined to the kidney. Microscopically, the tumor showed oncocytic tumor cells arranged in cords and nests. The tumor cells showed eosinophilic cytoplasm with monotonous round nuclei. Focally, the cells showed perinuclear halos but there were no wrinkled or double nuclei. Nucleoli were not prominent and no mitotic activity, vascular invasion or necrosis could be identified. (Fig. 1). The tumor cells were focally positive for CK7 and negative for CD10, vimentin, Ki-67, and Hale colloidal iron. On electron microscopy, the cytoplasm of tumor cells was packed with mitochondria and some cells contained abundant lipofuscin granules. The case was reviewed by 2 expert GU pathologists who concluded that the tumor showed characteristics of both OC and chrcc. One pathologist favored an OC and the other designated it as a renal oncocytic neoplasm. Molecular Studies Given the uncertainty in diagnosis, a virtual karyotype was performed with a 250K Nsp SNP Microarray (Affymetrix, Santa Clara, CA) as described before. 6 The results showed loss of chromosomes 1 and 19 (Fig. 2). The pattern of chromosomal gain/loss was compared with patterns of genomic changes observed in 26 OCs and 16 chrccs (Fig. 3). The pattern obtained for this tumor matched the pattern seen in OCs: either normal diploid complement, or complete/partial loss of chromosome 1 in 50% of cases. DISCUSSION Renal cell tumors are unique among solid tumors in that each subtype has characteristic recurrent chromosomal abnormalities. 7 11 These genetic changes are specific for each of the morphologic variants of renal cell tumors. More than 98% of clear cell RCCs 20 www.pathologycasereviews.com Pathology Case Reviews Volume 15, Number 1, January/February 2010

Pathology Case Reviews Volume 15, Number 1, January/February 2010 Molecular Approach to Oncocytic Renal Tumors FIGURE 1. Morphologic features of an oncocytic neoplasm. A, Oncocytic neoplasm with tumor cells arranged in cords and nests, note lack of loose stroma typically seen in oncocytomas (40 ). B, 100 view of tumor cells showing eosinophilic cytoplasm with monotonous round nuclei. C and D, 200 views showing presence of perinuclear clearing and areas with discohesive cells. FIGURE 2. Whole genome view of virtual karyotypes from oncocytic neoplasm. The uppermost plot represents the estimated copy number as a log 2 ratio averaged over 30 SNPs; middle bar represents heterozygous call bars. Bottom plot represents allelic differences between low and high intensity alleles (for heterozygous SNPs only). Note loss of chromosomes 1 and 19 (arrows). (ccrcc) show deletions in the short arm of chromosome 3. 5,8,10 Papillary RCCs (prcc) usually display trisomies of chromosomes 7 and/or 17 with or without loss of chromosome Y. 12 A hypodiploid chromosomal complement, with monosomies of chromosomes 1, 2, 6, 10, 13, 17, and 21, characterizes chrcc 4 ; and OC can show a normal diploid karyotype or copy number alterations in chromosomes 1 and Y, along with translocations involving 11q13. 13,14 Given the recurrent nature of these chromosomal imbalances, several pathologists have suggested using them as a diagnostic aid in renal tumors. 4,8,15 However, this knowledge has not been consistently applied in the routine diagnostic evaluation of renal neoplasms. Immunohistochemistry (IHC) is a reliable method for identification of common renal cell tumor variants; however, OC and chrcc often show overlapping immunohistochemical profiles. 3,16 This is not surprising given that OC and chrcc have almost indistinguishable gene expression profiles. 17 Cytokeratin 7 (CK7) has been reported to be a marker for chrcc but recent studies have reported strong CK7 staining in up to 27% of OC and focal staining in a higher percentage. 18,19 Recently, new markers to distinguish chrcc and OC have been reported, including S100A, CD82 (Kangai 1 KAI-1 ) and Claudins 7/8 (Table 1). 20 23 These markers show differential staining between classic chrcc and classic OC. Most of these studies, however, have not evaluated the performance of these markers on panels that included cases of eosinophilic chrcc and unclassified oncocytic neoplasms. These latter groups are the categories in which this differentiation is most needed. It should also be noted that performance of these markers in classic chrcc cases does not necessarily translate to performance in cases of tumors with noncharacteristic morphology since protein expression patterns could be different between classic chrcc and its eosinophilic variant. As with other IHC stains, it is possible that panels composed of these new markers could give better sensitivity and specificity than single markers but this has not yet been explored. Molecular methods to evaluate chromosomal abnormalities have recently emerged as a reliable alternative to IHC and conventional cytogenetics to evaluate oncocytic renal tumors. 4,15,24 Brunelli et al have shown that chromosomal loss profiles are similar between classic and eosinophilic chrcc and that fluorescence in situ hybridization (FISH) is a useful approach to differentiate the latter from OC. 4 However, multiple FISH probes are required to differentiate these 2 tumors, significantly increasing the cost associated with this testing. Furthermore, panels that can help with the differential diagnosis of all oncocytic renal tumors would require even more probes. Recently, microarray-based tools that provide high-resolution, genome-wide assessment of tumor genomes and reveal chromosome copy numbers have been developed. These array-based copy number platforms, like array-based comparative genomic hybridization (acgh) or SNP arrays can be employed to detect chromosomal imbalances in renal cell tumors. 10,15,25,26 2010 Lippincott Williams & Wilkins www.pathologycasereviews.com 21

Monzon et al Pathology Case Reviews Volume 15, Number 1, January/February 2010 FIGURE 3. Cumulative frequency of chromosomal lesions in Oncocytoma and Chromophobe Renal Cell Carcinoma. Gains are indicated as positive values (light gray) and losses as negative values (dark gray). TABLE 1. Novel Candidate Protein Markers for Distinction of Oncocytoma and Chromophobe RCC Positive Staining Eosinophilic chrcc Included Marker Oncocytoma Chromophobe RCC in Study Reference Caveolin-1 100% (21/21) 12% (3/26) Yes, showing same pattern as Garcia and Li 19 classic chrcc CD82 (Kangai 1, KAI-1) 2% (2/28) 87% (27/31) Not specified Kauffman et al 22 Claudin 7 23% (10/43) 67% (24/36) Not specified Hornsby et al 23 Claudin 8 88% (15/17) 27% (3/11) Not specified Onsunkoya et al 21 S100A 93% (37/40) 6% (2/51) Yes, 3 eosinophilic chrcc evaluated, only 1 positive Rocca et al 20 RCC indicates renal cell carcinoma; chrcc, chromophobe RCC. In this report, we used chromosomal analysis with SNP arrays (also called virtual karyotyping) as an ancillary tool for the diagnosis of a challenging case of an oncocytic renal neoplasm that could not be resolved by light and electron microscopy. The differential diagnosis for oncocytic renal tumors includes a heterogeneous group of neoplasms ranging from benign to highly malignant tumors such as OC, clear cell RCC with granular cells, chrcc, and type 2 prcc, as well as epithelioid angiomyolipoma. 2,27 In a recent study, we reported that oncocytic tumors accounted for 28% of renal neoplasms with difficult morphology that generated diagnostic discrepancies among a panel of expert GU pathologists. 6 Although not included in the 2004 WHO classification, 1 the terms granular cell RCC, oncocytic RCC/neoplasm, and eosinophilic RCC/neoplasm are commonly but incorrectly used as diagnostic entities. This is probably due to the fact that usage of the term unclassified renal cell carcinoma carries significant prognostic implications that do not apply to the majority of nonclassifiable oncocytic renal tumors. 28 However, since these diagnoses do not correspond to a recognized entity, they do not provide adequate information for patient management decisions. Thus, efforts to classify these tumors are important in determining appropriate patient care. In the case presented herein, the morphologic and immunohistochemical features had narrowed the differential diagnostic possibilities for this oncocytic tumor to OC versus chrcc. The distinction between these 2 entities is important, since one of them is a benign neoplasm and the other a malignant tumor with metastatic potential. As shown in Figure 3, the genomic profiles from OC and chrcc are quite different, with chrcc showing multiple chromosomal losses which allow distinction from OC s profile. Figure 2 shows the virtual karyotype from the current reported case showing losses only in chromosomes 1 and 19, which is a pattern consistent with that observed in OCs. In 2 recent studies, we showed the utility of virtual karyotyping which allowed molecular classification of renal tumors exhibiting difficult/challenging morphology. 5,6 These combined studies provided a total of 43 cases of morphologically challenging tumors of which 22 (51%) were tumors with oncocytic/eosinophilic features. This latter group of tumors included cases that represented the most common diagnostic dilemmas for renal oncocytic neoplasms: OC versus chrcc, ccrcc with granular cells versus eosinophilic chrcc and unclassified oncocytic tumors. Virtual karyotyping was able to establish a diagnosis based on patterns of chromosomal losses in 20 cases (90%). Two cases were not able to be classified because they showed novel genomic patterns which have not been reported in any of the known RCC morphologic subtypes. 5,6 Apart from the oncocytic tumors, virtual karyotyping was successful in classifying 41 of the 43 cases of morphologically challenging tumors (95%). This highlights the advantage of a genome-wide 22 www.pathologycasereviews.com 2010 Lippincott Williams & Wilkins

Pathology Case Reviews Volume 15, Number 1, January/February 2010 Molecular Approach to Oncocytic Renal Tumors method that can be used for all difficult renal tumors, rather than a focused approach, such as FISH, that would necessitate different probes for each of the tumors in the differential diagnosis. As mentioned above, there are 2 widely used techniques for genome-wide scanning of chromosomal aberrations in human tumors: array CGH, 25 and SNP arrays. 15 Array CGH has been used to accurately classify RCCs based on the specific genetic alterations as described above 10,25 ; however, array CGH cannot detect regions of copy neutral loss of heterozygosity (LOH) or acquired uniparental disomy (aupd). This genetic lesion, in which 2 copies of the same chromosome or chromosomal region are present, has been reported to constitute 50% to 80% of the LOH in human cancers. 29 32 SNP arrays not only provide chromosome copy number data but also provide genotypes which can be used to determine regions of LOH that would go undetected by acgh and FISH. Although the clinical significance of copy neutral LOH has not been well established, a recent study showed that copy neutral LOH in17p was associated with homozygous mutations in TP53 and conferred significantly shorter survival times for patients with glioblastoma multiforme. 33 It is also important to understand the limitations of array-based technologies. Both acgh and SNP arrays are unable to detect tetraploidy with certainty (although certain features of virtual karyotypes with SNP arrays can suggest tetraploidy), 34 and also fail to detect inversions or balanced translocations. In the context of renal tumors, this indicates that virtual karyotyping cannot be used to identify the presence of translocations involving Xp11.2 or t(6;11 p21;q12), which are the genetic lesions underlying the MiTF/TFE family of translocation renal tumors. 35,36 CONCLUSION Correct classification of renal tumors is critical for an accurate diagnosis and for subsequent correlation of prognostic information with appropriate patient management. This would include consideration for eligibility in clinical trials of new targeted therapies. 37 Thus, the ability to clearly identify renal tumor subtypes has significant implications with respect to patient care. Renal cell tumor subtypes can be reliably distinguished by their patterns of chromosomal gains/losses. 10,15,38 Virtual karyotyping with SNP arrays has proven to be a useful ancillary study for the diagnosis of morphologically challenging renal tumors, including oncocytic renal neoplasms. 5,6 We have presented a case in which conventional approaches with morphology, immunohistochemistry and electron microscopy were not able to unequivocally classify an oncocytic renal tumor. Virtual karyotyping was able to identify a pattern of chromosomal imbalances consistent with an OC. This case highlights the utility of novel molecular tools in the diagnosis of renal cell neoplasms. REFERENCES 1. Eble JN, WHO, IARC, IAP. Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs. Lyon, France: IARC Press, Oxford University Press (distributor); 2004. 2. Abrahams NA, Tamboli P. Oncocytic renal neoplasms: diagnostic considerations. Clin Lab Med. 2005;25:317 339. 3. 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Monzon et al Pathology Case Reviews Volume 15, Number 1, January/February 2010 32. Kloth JN, Oosting J, van Wezel T, et al. Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex genetic alterations in cervical cancer. BMC Genomics. 2007;8:53. 33. Yin D, Ogawa S, Kawamata N, et al. High-resolution genomic copy number profiling of glioblastoma multiforme by single nucleotide polymorphism DNA microarray. Mol Cancer Res. 2009;7:665 677. 34. Lo KC, Bailey D, Burkhardt T, et al. Comprehensive analysis of loss of heterozygosity events in glioblastoma using the 100K SNP mapping arrays and comparison with copy number abnormalities defined by BAC array comparative genomic hybridization. Genes Chromosomes Cancer. 2008;47: 221 237. 35. Argani P, Ladanyi M. Translocation carcinomas of the kidney. Clin Lab Med. 2005;25:363 378. 36. Camparo P, Vasiliu V, Molinie V, et al. Renal translocation carcinomas: clinicopathologic, immunohistochemical, and gene expression profiling analysis of 31 cases with a review of the literature. Am J Surg Pathol. 2008;32: 656 670. 37. Terrone C, Volpe A. The role of pathology for clinical decision-making in renal cell carcinoma is increasing. Eur Urol. 2007;51:1166 1168. 38. Furge KA, Lucas KA, Takahashi M, et al. Robust classification of renal cell carcinoma based on gene expression data and predicted cytogenetic profiles. Cancer Res. 2004;64:4117 4121. 24 www.pathologycasereviews.com 2010 Lippincott Williams & Wilkins