Clinical genomics of renal epithelial tumors

Cancer Genetics 204 (2011) 285e297

REVIEW

Clinical genomics of renal epithelial tumors Jill M. Hagenkord a,b, Zoran Gatalica c, Eric Jonasch d, Federico A. Monzon e,f,* a b

Department of Pathology, Creighton University School of Medicine, Omaha, NE, USA; iKaryos Diagnostics, Inc., Palo Alto, CA, USA; c Caris Life Sciences, Inc., Phoenix, AZ; d Department of Genitourinary Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA; e Department of Pathology and Laboratory Medicine, The Methodist Hospital and The Methodist Hospital Research Institute, Houston, TX, USA; f Department of Pathology, Weill-Cornell Medical College, New York, NY, USA Kidney and upper urinary tract cancers account for approximately 54,000 cases every year in the United States, and represent about 3.7% of adult malignancies, with more than 13,000 annual deaths. Classification of renal tumors is typically based on histomorphologic characteristics but, on occasion, morphologic characteristics are not sufficient. Each of the most common histologic subtypes harbors specific recurrent genetic abnormalities, such as deletion of 3p in conventional clear cell carcinoma, trisomy 7 and 17 in papillary renal cell carcinoma, multiple monosomies in chromophobe renal cell carcinoma, and a nearly diploid genome in benign oncocytomas. Knowledge of this information can provide diagnostic support and prognostic refinement in renal epithelial tumors. Identification of the specific subtype of a renal tumor is critical in guiding surveillance for recurrence and the appropriate use of targeted therapies. Cytogenomic arrays are increasingly being used as a clinical tool for genome-wide assessment of copy number and loss of heterozygosity in renal tumors. In addition, the improved understanding of the hereditary causes of renal tumors and their role in sporadic malignancies has led to the development of more effective targeted therapies. This review summarizes the genetic and genomic changes in the most common types of renal epithelial tumors and highlights the clinical implications of these aberrations. Keywords Renal cell carcinoma, diagnosis, prognosis, genetics, chromosomal imbalances, cytogenomic array, cytogenetics, SNP array, virtual karyotype ª 2011 Elsevier Inc. All rights reserved.

Kidney and upper urinary tract cancers account for approximately 54,000 cases every year in the United States, and represent about 3.7% of adult malignancies, with more than 13,000 annual deaths (1,2). Renal cell carcinoma (RCC) is the most common renal malignancy, with three common subtypes representing about 95% of all renal tumors: clear cell (ccRCC, 75% of RCC), papillary (pRCC, 10%), and chromophobe (chRCC, 5%). The fourth most common renal epithelial tumor is oncocytoma (OC, 5%), a benign neoplasm (Figure 1) (3). Although the majority of renal tumors occur in a sporadic fashion, approximately 2e4% occur in the setting of hereditary predisposition syndromes (4). There are four main hereditary cancer syndromes involving the renal epithelium: von Hippel Lindau (VHL) syndrome, which predisposes to development of ccRCC; hereditary papillary  syndrome renal cell carcinoma (HPRCC); Birt-Hogg-Dube

Received June 9, 2011; accepted June 10, 2011. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergen.2011.06.001

(BHDS), which predisposes to development of chRCC and OC; and hereditary leiomyomatosis and renal cell carcinoma (HLRCC), which predispose to pRCC and collecting duct carcinomas (3). Categorization of renal tumors can typically be done on the basis of histomorphologic characteristics. The correct determination of histologic subtype is critical because each has a distinct biologic behavior and therapeutic indications (5e7). However, morphologically challenging cases do occur. Even with generous sampling of large resection specimens, some tumors may have non-specific characteristics or overlap in morphologic features between tumor types. These morphologically challenging tumors are usually termed “unclassified renal cell carcinomas,” “renal cell carcinoma not otherwise specified,” or “eosinophilic renal carcinoma” in surgical pathology reports (3). Non-specific diagnoses confound efforts of the clinical team to predict tumor behavior, define appropriate follow up strategies, and guide therapeutic decisions. Difficulty in the morphologic assessment of renal tumors is compounded when evaluating scant tissue obtained from biopsy, or when architectural

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Figure 1 Morphology and genomic profiles for the most common renal epithelial tumors. Each renal epithelial tumor has morphologic (left column) and chromosomal copy number profiles (right column) that are characteristic to each subtype (red, loss; blue, gain; red stripes, aUPD). (A) Clear cell RCC, n Z 130, with characteristic loss of 3p and frequent imbalances in chromosomes 5, 7, 9, and 14. (B) Papillary RCC, n Z 26, with characteristic gain of chromosomes 7 and 17 and frequent imbalances in chromosomes 3 (including aUPD), 12, 16, and 20. (C) Chromophobe RCC, n Z 18, note hypodiploid complement with frequent losses of chromosomes 1, 2, 6, 10, 13, 17, and 21. (D) Oncocytoma, n Z 30, with majority of tumors showing normal chromosomal complement and frequent complete or partial loss of chromosome 1.

context is lost, as in fine needle core biopsy specimens (8,9). Immunohistochemical (IHC) profiles can help categorize morphologically challenging tumors (10), but sometimes overlapping phenotypes or non-specific staining profiles render IHC unsuccessful in resolving the differential diagnostic dilemmas. Other laboratory studies, such as conventional cytogenetics, cytogenomic arrays, fluorescence in situ hybridization (FISH), or microsatellite polymerase chain reaction to assess loss of heterozygosity (LOH) can further aid in classification. These assays are able to assess copy number changes directly or by inference (LOH), either in a global or targeted approach. They are useful for classification of renal epithelial tumors because each of the most common subtypes harbors specific recurrent genomic abnormalities, as described below (Table 1, Figure 1) (11). Each assay has inherent strengths and weaknesses that should be considered when evaluating published data and when selecting the appropriate methods for clinical diagnostics (12). Cytogenomic arrays, such as array comparative genomic hybridization (aCGH) and single-

nucleotide polymorphism (SNP) arrays, have been used to classify RCC on the basis of the specific genomic profiles for each subtype (Figure 1) (13,14). Cytogenomic arrays can provide not only diagnostic support, but also identify additional genomic changes, some of which are associated with outcome in specific subtypes. Although most patients with renal epithelial tumors have an excellent prognosis, 30% of patients with initially organconfined ccRCC will develop metastases. In addition, due to the paucity of overt clinical manifestations of localized disease, up to 30% of patients will present with metastases at the time of initial diagnosis (2). Treatment for advanced or metastatic kidney cancer is a formidable challenge with the traditional therapies currently available. Investigation of the Mendelian single-gene syndromes, such as von Hippel Lindau syndrome (VHL: VHL gene), hereditary papillary renal  (BHD: cell carcinoma (HPRCC: MET gene), Birt-Hogg-Dube BHD gene), and hereditary leiomyomatosis renal cell carcinoma (HLRCC: FH gene), however, have provided an opportunity to develop pathway-specific therapies (6).

Clinical genomics of renal tumors Table 1

287

Frequency of classic chromosomal aberrations in renal epithelial neoplasms

Type of renal tumor

Classic cytogenetic findings

Clear cell RCC

del(3)(p): 3p14, 3p21, 3p25-p26

Papillary RCC

Trisomy 7 and/or 17

Chromophobe RCC

Loss of 1, 2, 6, 10, 13, 17 and/or 21

Mucinous tubular and spindle cell carcinoma Oncocytoma

Loss of 1, 14, and 15 Chr 1 loss or normal

% Cases with chromosomal abnormality 98 98 81 100 100 67/43 100/38 100 100/50 95 74 100 100 100 100 100

N

Platform

52 118 26 11 98 19/20 9/16 6 19 10 19 4 12 6

LOH CG aCGH FISH SNP array FISH CGH FISH SNP array LOH FISH aCGH SNP array SNP array

10 15

FISH SNP array

Subtype

Low /high grade Type 1/type 2 Type 1/type 2

Reference (25) (26) (29) (28) (27) (119) (120) (28) (27) (25) (73) (29) (27) (27) (73) (27)

Abbreviations: N, number; CG, cytogenetics.

The aim of this review is to summarize our current knowledge on the genetics of the most frequent sporadic and hereditary renal epithelial tumors and to describe how evaluation of these abnormalities can help refine diagnoses, indicate prognoses, and potentially tailor therapy in patients with these tumors.

Clear cell renal cell carcinoma Histologic features of clear cell renal carcinoma ccRCC is the most common renal malignancy. It is believed to originate from cells in the proximal tubule of the nephron. Histologically, ccRCC may have a solid, alveolar, or acinar cell architectural pattern, and they typically contain a regular network of thin-walled blood vessels (Figure 1A) (3). The cytoplasm is commonly filled with lipids and glycogen, which dissolve upon histologic processing and create a clear cytoplasm surrounded by distinct cell membranes, which gives this tumor its name. There may also be a population of cells with eosinophilic cytoplasm, particularly in higher-grade tumors, which can cause a histopathologic dilemma about proper classification.

and is the leading cause of mortality. Approximately 80% of individuals with VHL syndrome have an affected parent, and about 20% have de novo gene mutations. VHL mutations are highly penetrant, and almost all individuals who have a mutation express disease-related symptoms by age 65. The manifestations and severity are highly variable both within and between families, even among those with the same mutation. Approximately 72% of the inherited mutations are point mutations or small deletions/insertions that can be detected by sequence analysis of all three exons of the VHL gene, and the remaining 28% are partial or complete gene deletions that can be detected by duplication/deletion analysis (16,17). The second hit can result from wild-type VHL gene deletion (i.e., 3p loss in the tumors), point mutations, or hypermethylation of the VHL promoter (18). Acquired mutations in the VHL gene are commonly found in sporadic ccRCC tumors (19,20). Non-VHL related familial ccRCC has also been reported. Some of these cases have been explained by recurrent chromosomal translocations involving 3p, while a few others arise in patients with BHDS (see below; (21e24)). However, a large number of patients with familial ccRCC have no discernible genetic cause (22).

Hereditary clear cell renal cell carcinoma

Genetics of sporadic clear cell renal cell carcinoma

Von Hippel Lindau disease (VHL) is an autosomal dominant cancer predisposition syndrome due to a germline mutation in the VHL tumor suppressor gene at chromosome 3p25-p26 (chr3:10,183,319-10,193,744/hg19). Patients with VHL have an autosomal dominant predisposition to develop ccRCCs, as well as hemangioblastomas of the brain, spinal cord, and retina, pheochromocytomas, pancreatic cysts, and endolymphatic sac tumors of the inner ear (3,15). Clear cell renal cell carcinoma occurs in about 40% of individuals with VHL

Like the VHL-related ccRCC, the vast majority of sporadic ccRCCs have a loss of chromosomal material on the short arm of chromosome 3 (Table 1) (25e29). The deletion of 3p can encompass the entire chromosomal arm or a minimally deleted region from 3p21 to 3p25, which includes the VHL tumor suppressor gene. The VHL protein targets hypoxiainducible factors (HIF) for ubiquitin-mediated degradation (7). When VHL is mutated, HIF accumulates in the nucleus, leading to increased transcription of a number of

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downstream pathway genes that promote angiogenesis, growth, and survival, such as vascular endothelial growth factor (VEGF), the glucose transporter (GLUT1) and transforming growth factor-beta (TGFB1) (6). Other recurrent chromosomal imbalances also occur in these tumors, with variable frequencies; some of the more common are trisomy 5 or 5q gain, trisomy 7, 9p loss, and 14q loss (Figure 1). Mutations in other genes located in 3p have been reported recently in sporadic tumors. PBRM1 (3p21.1, chr3:52,579,36852,713,739/hg19) appears to be mutated in approximately 40% of ccRCCs, while SETD2 (3p21.31, chr3:47,057,90047,205,467/hg19) seems to be mutated in approximately 3% (30,31). Both genes are involved in chromatin remodeling at the level of histone H3 modification and recognition. The biologic and clinical significance of these mutations is currently unknown.

Clinical relevance of genomics in clear cell renal cell carcinoma Genomic information can be used for diagnostic, prognostic, and predictive purposes in ccRCC. In morphologically ambiguous cases, identification of a deletion of 3p can help to clarify the diagnosis (27). However, monosomy and acquired uniparental disomy (aUPD) of chromosome 3 occasionally can be seen in pRCC and chRCC (Figure 1), so 3p loss should be evaluated in the context of morphology and, preferably, the complete genomic profile. Surgical excision is curative therapy in the majority of ccRCC cases, but up to 30% of patients with organ-confined tumors (stage pT1-2) develop metastases after nephrectomy (32). Nuclear grade has been shown to be the strongest morphologic predictor of poor outcome in ccRCC, but some studies indicate that it is not independent from overall stage (33e35). Specific chromosomal imbalances in ccRCC have Table 2

Genetic and genomic features associated with prognosis in clear cell renal cell carcinoma

Prognosis

Prognostic finding

Good

5q gain ccA gene expression profile 8q gain 9p loss

Poor

been associated with favorable outcomes or poor prognosis (Table 2). Gain of 5q has been reported as a good prognostic factor, while 8p gain, 9p loss, and 14q loss have been associated with poor outcome (25e27,29,36e40). Most of these associations, however, have been reported in small cohorts, except for 9p loss, whose association with poor outcome has been repeatedly observed in multiple studies (25,26,36,38,39). It is possible that these chromosomal imbalances might identify specific subsets of ccRCC patients, as it was recently shown that 9p loss appears to be specifically associated with poor outcome in patients with localized, small renal masses, and 14q loss seems to confer poor prognosis in patients with non-metastatic tumors (39,40). Prognostic categories based on gene expression or cytogenomic array profiling have also been reported. Brannon and coworkers described two subtypes of ccRCC (ccA/ ccB) based on gene expression profiling, with markedly different outcomes (41). In addition, Arai and coworkers reported that hierarchical clustering of ccRCCs based on patterns of chromosomal imbalances can identify two groups of patients with distinctly different recurrence-free survival and overall survival (OS; (42)). Understanding the genetic alterations in ccRCC and their impact on the VHL-HIF pathway has led to the development of targeted drugs for ccRCC patients with metastatic disease. Many of the HIF-regulated genes are targets for new therapeutic approaches for ccRCC, such as bevacizumab, sunitinib, sorafenib, and pazopanib (6,7). Bevacizumab, an antibody targeting VEGF-A, was shown to improve progression-free survival (PFS) in two randomized studies comparing bevacizumab plus interferon-alpha (IFN) to IFN monotherapy (43). Sorafenib, an oral small-molecule protein kinase inhibitor effective against the VEGF receptor (VEGFR), the platelet-derived growth factor receptor, and others, was approved in 2005 by the U.S. Food and Drug Administration (FDA) for treatment of metastatic ccRCC (44). It was closely

% Cases with feature

N

Platform

Reference

Association

(26) (41) (40) (26) (36) (25) (38)

Better overall survival (P Z 0.001) Better disease specific survival (P Z 0.002) Decreased overall survival (P < 0.0001) METs at Dx (P Z 0.006) 5y-CSS 43 vs. 88 (P < 0.001) Low vs. high stage Decreased survival (P Z 0.033 in multivariate analysis) Shorter recurrenceefree survival (P < 0.01) and higher likelihood of recurrence in pT1a tumors (P Z 0.01) Low vs. high stage Low vs. high grade (P Z 0.0143) Higher risk for recurrence (P Z 0.002) and shorter overall survival (P Z 0.030) in non-metastatic ccRCC Worse disease specific survival (P Z 0.002)

56.8 46

118 177

13 21 18 23 16

85 118 73 43 282

CG Gene expression microarray SNP array CG FISH LOH CG

13.8

703

CG / FISH

(39)

LOH aCGH SNP array

(25,29) (25,29) (40)

Gene expression microarray

(41)

14q loss

31 38 55

52 26 85

ccB gene expression profile

34

177

Abbreviations: N, number; CG, cytogenetics; Dx, diagnosis; CSS, cancerespecific survival.

Clinical genomics of renal tumors followed by sunitinib, which was approved in early 2006 (45). Pazopanib, another multiple kinase inhibitor, was approved in 2009 (46). These agents all provide a significant prolongation of PFS when compared to IFN therapy or to placebo, but it has been harder to confirm an OS advantage, possibly due to the crossover (change to a new therapy) to other effective agents on most of these clinical trials. The challenge at this point in time is to define tumor and host features responsible for innate and acquired resistance to anti-VEGF therapy, since no reliable predictive biomarkers have been identified to date. Our group recently reported that 8q gain seems to separate patients treated with anti-angiogenic agents into different subgroups with markedly different OS (40).

Papillary renal cell carcinoma Histologic features of papillary renal cell carcinoma Papillary renal cell carcinomas comprise approximately 10% of renal cell carcinomas (3). From a histological standpoint, pRCCs are characterized by tumor epithelial cells forming various proportions of papillae and tubules (Figure 1). The tumor papillae contain a delicate, fibrovascular core and can show aggregates of foamy macrophages (3). Papillary renal cell tumors have been morphologically divided into small (type 1) and large (type 2) cell tumors. Type 1 pRCC have papillae covered by small cells with scanty cytoplasm, arranged in a single layer on the papillary basement membrane. Type 2 pRCC tumor cells are typically of a higher grade with eosinophilic cytoplasm and pseudo-stratified nuclei on papillary cores (Figure 1) (3).

Hereditary papillary renal cell carcinoma Hereditary papillary renal cell carcinoma (HPRCC) is a highly penetrant hereditary renal cancer syndrome in which affected individuals are at risk for the development of bilateral, multifocal, type 1 pRCC (6,47). The kidney is the only organ known to be affected in HPRCC patients, and although the tumors are usually well differentiated, they are malignant and can metastasize. Unlike VHL, HPRCC involves the activation of a protooncogene, not inactivation of a tumor suppressor gene. HPRCC is due to missense mutations of the MET protooncogene at 7q31.2 (chr7:116,312,459-116,438,439/hg19), which results in the constitutive activation of the tyrosine kinase domain (18). The MET gene encodes the cell surface receptor for hepatocyte growth factor (HGF), also belonging to the tyrosine kinase class of receptor proteins (48), and MET gain-of-function mutations trigger cell proliferation, neovascularization, and increased cell motility (18). Molecular targeting approaches are being developed to inhibit the interaction of HGF and its receptor, and suppression of the downstream signaling cascade of activated c-MET (49). Type I pRCC can also be seen in BHDS (described further below). Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is another hereditary cancer syndrome that may manifest with pRCC. HLRCC is an autosomal dominant disorder characterized by cutaneous leiomyomata, uterine leiomyomata in females, and/or a single renal tumor. Most renal

289 tumors are type 2 pRCC, but other types of aggressive renal carcinomas can be seen (50,51). Penetrance for pRCC is lower than for the cutaneous and uterine manifestations, with only 20e35% of patients developing pRCC (6). Affected individuals are typically born with a heterozygous mutation in the FH gene at 1q42-q44 (chr1:241660906-241683054/ hg19), encoding the enzyme fumarate hydratase (52). The majority of mutations in FH are point mutations or small deletions/insertions (50,51), although exonic and whole-gene deletions have been reported (53,54). FH is an essential enzyme for the conversion of fumarate to malate in the Krebs cycle. The loss of FH function impairs the Krebs cycle, giving way to glycolitic metabolism, cellular pseudo-hypoxia, and up-regulation of HIF and HIF-inducible transcripts (6,55). The diagnosis of HLRCC is clinical, based on the presence of multiple cutaneous leiomyomas or by a single cutaneous leiomyoma in the presence of a positive family history of HLRCC-related tumors. The diagnosis is confirmed by testing of fumarate hydratase enzyme activity in cultured skin fibroblasts or lymphoblastoid cells showing reduced activity (
60%), or by molecular genetic testing (56). Studies of the tricarboxylic acid cycle and the VHL-HIF pathways have provided the foundation for metabolic therapeutic approaches in patients with HLRCC-associated kidney cancer as well as other hereditary and sporadic forms of RCC (7). In an interesting parallel to the HLRCC kindreds described above, germline mutations in subunits of the succinate dehydrogenase (SDH ) gene, another Krebs cycle enzyme, can predispose to early-onset kidney cancers in addition to paragangliomas and carry implications for medical surveillance (57,58). There have been reports of chromophobe RCC in patients with germline mutations in SDHB (hereditary paraganglioma/pheochromocytoma) (7,57,59).

Genetics of sporadic papillary renal cell carcinoma Mutations of MET have also been identified in a subset of tumors from patients with sporadic type 1 pRCC (7). The genomic profile of pRCC displays whole chromosomal gains, characteristically trisomy 7, which includes the HGF and MET genes, and trisomy 17. Gains of chromosomes 12, 16, and 20 are also frequently seen (Figure 1) (27,60). It has been proposed that renal papillary adenomas have only trisomy 7 and 17, while carcinomas are marked by additional numerical chromosomal changes (60). Trisomy 7 and 17 occur in both type 1 and type 2 pRCC (27,61), and debates persist in the literature regarding the validity of dividing pRCC into cytomorphologic subtypes. Type 1 pRCC, however, has been shown to have significantly better survival than type 2 (18,33,61).

Clinical relevance of genomics in papillary renal cell carcinoma RCC with papillary features and extensive clear cell changes may be difficult to classify as either ccRCC or pRCC. Xp11.2 translocation carcinoma (see below) also shows papillary architecture with clear cell features and is therefore usually within the differential diagnosis. Positive IHC staining for alpha-methylacyl-CoA-racemace and/or the presence of 7/17 gains can help make the diagnosis of pRCC in these

290 cases (62). Subtyping of pRCC is clinically important because tumor type is an independent predictor of outcome and the 5-year cancer-specific survival of pRCC is considerably better than that of ccRCC in the non-metastatic setting. In addition, the presence of 1p gain in pRCC has been reported as a poor prognostic factor (63). Unfortunately, once metastatic, the prognosis of pRCC is poor, and several studies suggest it is inferior to ccRCC (64). This is due, in part, to the lack of effective systemic therapies for this histologic subtype. Targeting the consequences of HIF up-regulation and reversing or modulating the inactivation of FH are potential molecular therapeutic targets for treatment of metastatic pRCC arising in the context of HLRCC (6). For patients with MET mutations or patients with sporadic pRCC, clinical trials targeting the MET pathway are currently underway. Other clinical trials have demonstrated that the mammalian target of rapamycin inhibition may be of some value (65), as is the epidermal growth factor blockade (66). Linkage between specific mutations or chromosomal patterns and response to specific agents may help refine therapy for these patients in the future (7).

Chromophobe renal cell carcinoma Histologic features of chromophobe renal cell carcinoma Chromophobe renal cell carcinoma (chRCC) accounts for approximately 5% of surgically removed renal epithelial tumors (3). From a histological standpoint, chRCC shows solid growth pattern with focal calcifications and broad fibrotic septae. The tumor cells are large, polygonal, with clear cytoplasm and prominent cell membranes. These cells are commonly admixed with smaller cells with granular, eosinophilc cytoplasm. In contrast to ccRCC, the blood vessels tend to be thick-walled and eccentrically hyalinized (3). Oncocytic or eosinophilic variants of chRCC are not uncommon and represent a diagnostic challenge by morphology (67).

Hereditary chromophobe renal cell carcinoma Birt-Hogg-Dube syndrome (BHDS) is an autosomal dominant condition characterized clinically by benign cutaneous tumors (fibrofolliculomas, trichodiscomas, and acrochordons), pulmonary cysts resulting in recurrent spontaneous pneumothoraces, and multifocal, bilateral renal cancer of varying histologies (68,69). BHDS shows clinical heterogeneity, and patients do not always have the characteristic phenotypical triad (70,71). The most common renal cell tumor seen in BHDS is the so-called oncocytic hybrid tumor (50%), which is a hybrid of morphologic features between oncocytoma and chromophobe histologic cell types. Chromophobe RCC presents in 33% of BHDS patients, and renal oncocytoma in 5% (6). Other types of renal tumors reported with lower frequency include ccRCC and pRCC (3). Mutations in the folliculin gene (FLCN, chr17:17115529-17140502/hg19) are considered causative (72). FLCN mutations have been reported in 88% of BHDS family members and can be found in several exons of the gene, although there is a mutational hotspot in

J.M. Hagenkord et al. exon 11 (70). A common 3.5-megabase deletion of 17p11.2 is present in approximately 70% of renal tumors from individuals affected with BHDS, supporting the tumor suppressor gene function of FLCN (7,18).

Genetics of somatic chromophobe renal cell carcinoma Chromophobe RCC typically has a hypodiploid genome, with whole chromosomal losses of Y, 1, 2, 6, 10, 13q, 17, and 21 (Figure 1) (27,73). These chromosomal losses have been demonstrated by metaphase cytogenetics, FISH, CGH, SNP array analysis, and microsatellite LOH (27,73e75). To date, no recurrent single gene mutational events have been reported for sporadic chRCC (76).

Clinical relevance of genomics in chromophobe renal cell carcinoma ChRCC are rarely aggressive so correct diagnosis is critical for appropriate patient management. Most chRCC are stage T1 and T2 (86%), and only 10% show extension through the renal capsule (3). Chromophobe RCC demonstrates morphologic overlap with ccRCC, especially when showing eosinophilic cytoplasm (77). The characteristic pattern of chromosomal losses is helpful in establishing the correct diagnosis (67,73). ChRCC and hybrid chRCC/oncocytoma tumors can be seen in BHDS. The function of the FLCN gene product, folliculin, is not well-characterized, but it has been linked to regulation of the mTOR, AMPK, LKB1, and TSC1/TSC2 pathways (7,78). The finding that mutations of two additional Krebs cycle enzymes (FH and SDH ), and disruption of the nutrient sensing pathway via mutation of FLCN all cause renal cell carcinoma provide important clues to the pathogenesis of RCC and the role of metabolism in carcinogenesis. Future work on targeting and modulating the glycolytic pathway in RCC will likely pay dividends in the discovery of new agents for the treatment of chRCC, and possibly also ccRCC.

Oncocytoma Histologic features of renal oncocytomas Renal oncocytomas (OC) are benign neoplasms which account for about 3e7% of all renal tumors. Oncocytoma tumor cells have abundant, granular eosinophilic cytoplasm (reflecting an abundance of mitocondria) arranged in nests, tubulocystic, solid, or trabecular patterns (3). One of the most prominent microscopic features of oncocytoma is the edematous, myxomatous, or hyalinized stroma that surrounds the tumor cells (Figure 1D). The nuclei are typically round and centrally located, but it is common to observe cells with large, bizarre nuclei. In addition, nuclear halos (a feature usually associated with chRCC) may be seen in OC, sometimes making distinction from chRCC difficult (79).

Clinical genomics of renal tumors

Hereditary renal oncocytomas As discussed above, renal oncocytomas can occur in patients with BHDS. Renal oncocytosis is a disease in which patients have a propensity to develop multiple synchronous and metachronous oncocytic tumors in the kidneys. This presentation has been observed in the presence of BHDS, but it is also seen in cases without BHDS and in cases with chronic renal failure (80,81).

Genetics of somatic renal oncocytomas The majority of renal oncocytomas (50e60%) show a normal chromosomal complement (Figure 1) (27,73,75,82). Approximately 40% of oncocytomas show complete or partial loss of chromosome 1. Other frequent changes include loss of Y (15%) and monosomy 14 (15%), which are usually seen in conjunction with chromosome 1 loss. Trisomy 7 and structural rearrangements involving 11q12-q13 have been reported in a small percentage of OC. Chromosomal abnormalities seem to be more frequent in sporadic tumors, while familial OC appears to have a higher frequency of cytogenetically normal tumors (83). To date, no recurrent single-gene mutational events have been reported for sporadic OC.

Clinical relevance of genomics of renal oncocytomas Renal oncocytomas behave in a benign fashion. Although one case of metastatic oncocytoma has been reported, this tumor was characterized only by IHC and no molecular analyses were performed (84). Thus, the true nature of this “metastatic OC” has not been established with certainty, and might represent misclassified chRCC or the eosinophilic variant of ccRCC. The distinction of OC from chRCC is one of the diagnostic pitfalls frequently encountered with renal epithelial tumors (77). Genomic profiles can be useful in correctly distinguishing between benign and malignant renal tumors. For example, many studies have reported that chromophobe RCC shows complex simultaneous losses of chromosomes 1, 2, 6, 10, 13, 17, and 21 (27,73); and, although occasional losses of each of these chromosomes have been reported in OC, the simultaneous loss of all these chromosomes is only seen in chRCC.

Rare renal epithelial tumors Xp11.2 translocation renal cell carcinomas Renal cell tumors associated with an Xp11.2 translocation typically occur in children or young adults (3). The architecture of these tumors shows characteristic morphologic features, with cells presenting abundant clear cytoplasm arranged in nests, papillary or pseudopapillary structures, and frequent psammoma bodies (calcium deposits). These tumors are characterized by the presence of a chromosomal rearrangement in Xp11.2. This translocation involves a breakpoint at the TFE3 gene on Xp11.2 and various fusion partners, including PRCC (1q21), ASPL (17q25), PSF (1p34), CLTC (17q23), and NonO (Xq12) (18,85). Positive

291 nuclear immunostaining for TFE3 is a surrogate marker for an Xp11 translocation RCC when observed in pediatric tumors, but the sensitivity of this stain in adult tumors has not been established (86,87). These tumors have an unpredictable behavior, with some patients presenting with widely metastatic disease and others showing more indolent clinical course. Lymph node status is currently the best prognostic marker for this entity (88,89). It is expected that knowledge of the molecular pathways affected by TFE3 overexpression may help guide therapy, because it appears that at least some Xp11.2 translocation tumors are associated with defects in mitotic checkpoint control. This might render them more responsive to chemotherapeutic agents such as vincrinstine and paclitaxel (18,90).

Mucinous tubular spindle cell carcinoma Mucinous tubular and spindle cell carcinomas (MTSCC) are an uncommon, low-grade renal epithelial tumors characterized morphologically by small, elongated tubules lined by cuboidal cells and/or cords of spindled cells separated by mucinous stroma (91). Not all MTSCC have classic morphology, however, and some show overlaping features with pRCC (92,93). Genetically, these tumors are characterized by multiple chromosomal losses, but the exact losses described have varied between method and investigator (91). Several groups have reported complete or partial losses of 1, 4, 6, 8, 13, 14, 15, and 22 when using genome-wide methods (27,94,95). Compared with MTSCCs, other RCCs with spindle cell features (sarcomatoid change) behave aggressively, thus underscoring the importance of correct categorization of these tumor types. It has been suggested that MTSCC is a variant of papillary renal cell carcinoma, based on morphological and immunohistochemical similarities (91,96), although the chromosomal profiles are markedly different (27,97).

Discussion Genetic and genomic information can assist in the diagnosis, prognosis, and therapeutic management of renal epithelial tumors. Often, the diagnosis is straightforward for renal tumors, based on their classic histologic characteristics. A small but significant percentage of tumors, however, have ambiguous morphologic features, and approximately 5% are reported as “unclassified” due to overlapping morphologic characteristics, even in well-sampled nephrectomy specimens (3,98). Proper diagnosis is critical to ensure appropriate patient management because each subtype of renal epithelial tumor has a specific recurrence risk and treatment regimen. The 5-year survival and disease-free progression of renal cell carcinomas varies by subtype: chRCC (100 and 94%), pRCC (86 and 88%), ccRCC (76 and 70%), RCC unclassified (24 and 18%) (5). Oncocytomas are benign neoplasms with no risk of metastasis, but they can morphologically mimic some renal cell carcinomas and are often part of the differential diagnosis. Cytogenomic profiles obtained from array-based studies can be useful in the evaluation of diagnostically challenging renal epithelial tumors, even in cases where FISH and IHC yield ambiguous results (27,99).

292 Kim et al. reported that SNP arrays could resolve 94% of morphologically challenging renal tumors (27). In addition, Viera et al. reported that the genetic diagnosis of renal tumors by comparative genomic hybridization on FNA biopsies can improve differential diagnosis (98). Core biopsies (CB) or fine-needle aspirates (FNA) are now frequently used for diagnostic purposes and to guide appropriate intervention. CB/FNA sampling poses an additional diagnostic challenge due to scant tissue, sampling error, and loss of architectural context (8,9). These diagnostic modalities are being used more frequently because of the increased detection of incidental kidney tumors with abdominal CT and/ or ultrasonography from the evaluation of an unrelated problem (98). Surgical resection is generally indicated in patients with a solid renal mass to determine if the mass is malignant (100). Surgery, if performed, provides not only diagnostic tissue, but in most cases also definitive therapy. For the elderly and others at high surgical risk, surgery may be deferred if the tumor is radiologically benign-appearing or percutaneous radiofrequency ablation or cryotherapy may be an alternative. In these cases, CB or FNA are important to confirm the diagnosis prior to treatment (101). In this context, it is expected that evaluation of genomic features of renal epithelial tumors will assist in providing diagnostic and prognostic information in samples with limited tissue. In addition to assisting in correct classification, the ability to identify tumors within each subtype that are most likely to recur would allow management strategies to be further tailored to each patient’s risk. For ccRCC and pRCC, the strongest predictors of survival are tumor stage, nuclear grade, and necrosis (33,102,103). However, there is currently no reliable biomarker that can predict metastatic or local recurrence in patients with organ confined tumors (stages I and II), and the significance of histologic subtype for prognosis is not independent of stage (102,104). Cytogenomic profiles can assess the diagnostic genomic changes as well as genomic changes associated with prognosis. For example, detection of a 3p deletion in a morphologically challenging renal tumor would support the diagnosis of ccRCC because nearly 100% of ccRCC demonstrate this feature (Figure 1). In addition, a cytogenomic profile could detect the deletion of 9p or 14q, both associated with poor prognosis (Table 2) (26,29,36,37,105). This information could allow surgeons and oncologists to design appropriate monitoring and interventions for those tumors that are more likely to recur, such as changing monitoring practices or the use of adjuvant treatments. The emergence of targeted therapies, such as antiangiogenic drugs and mTOR inhibitors, has dramatically improved the progression-free survival (PFS) of patients affected by ccRCC, and is gradually improving OS. Understanding of the VHL-HIF-VEGF cellular hypoxia pathway has provided the foundation for the development of treatment strategies based on tyrosine kinase inhibitors (TKIs) with activity against VEGF receptors, such as sunitinib and sorafenib (Table 3) (7). Likewise, MET mutations are seen in hereditary and sporadic pRCC, and clinical trials targeting the MET pathway are ongoing (7). Preclinical studies are underway targeting the BHD gene pathway for BHDS and sporadic chRCC (7). At this moment, there is no evidence that mutational status on VHL or MET genes modifies a tumor’s response to targeted agents. However, it is possible that as

J.M. Hagenkord et al. more specific targeted drugs are developed, the evaluation of mutational status might be necessary to qualify for therapy. Assessing the cytogenomic profiles of renal epithelial tumors allows for diagnostic support, refined prognostic information, and can guide appropriate choice of therapy. There are several techniques in use in clinical and research laboratories to assess chromosome copy number and/or LOH status, including conventional cytogenetics, FISH analysis, microsatellite LOH analysis, and array-based genomic analysis (aCGH or SNP arrays). The great majority of diagnostic and prognostic alterations can be detected with any of these approaches. Each technique has strengths and limitations, however, and these differences may explain some of the discrepancies reported in the literature regarding frequency of chromosomal imbalances, and should be kept in mind when guiding clinical testing strategies and evaluating diagnostic results. Conventional cytogenetics is the method of choice when assessing for non-recurrent balanced translocations, such as those present in familial non-VHL ccRCC. FISH can be used to detect the presence of recurrent chromosomal translocations, such as those present in pediatric tumors involving the Xp11.2 locus. Array-based genomic analysis is quickly becoming the method of choice for comprehensive evaluation of chromosomal imbalances in kidney and other solid tumors. Variability in the frequency of chromosome copy number and LOH status in renal tumors reported in the literature may be due to limitations in different assays (Table 1). In addition, variability may also arise from improper data normalization in tumors with hyperdiploid or hypodiploid genomes when using array-based techniques. Most software programs for arraybased copy number analysis use a mean or median normalization step to adjust for differences in signal intensity between different samples. This approach assumes that the overall ploidy of the test genome is close to two. When the test genome is hypo- or hyperdiploid, the normalization process results in the test genome log2 ratios being adjusted by an incorrect factor. In the case of MTSCC or chRCC, the genome is hypodiploid and the default normalization will make the diploid regions appear to be triploid. Without corrections to the normalization process, CGH and array-based genomic analysis may yield false chromosomal gains (106). The rapid development of NGS technologies may allow the simultaneous analysis of mutational status, translocation events, and chromosome copy number changes with a single technology in the near future (107,108). Although not specifically applied to renal tumors, promising advances in copy number analysis with NGS have been shown to be useful in paraffin-embedded tissues and, thus, could enable the routine clinical utilization of this approach (109).

Conclusions The correct categorization of renal tumors has an immediate impact on diagnosis, prognosis, therapy, and post-surgical surveillance. Although the chromosomal lesions that characterize each subtype of renal epithelial neoplasms have been known for some time, this knowledge has not been incorporated in the routine diagnostic evaluation of these tumors. The advent of new technologies, such as cytogenomic arrays, enables the use of cytogenomic profiles in the

Relationship between genetic alterations and novel targeted approaches for treatment of renal cell carcinoma Targeted agents in clinical trials

Syndrome

Phenotype

Gene/location

Pathways affected

FDA-approved compounds

Von Hippel Lindau Disease (110)

Kidney  Renal cysts  Mulifocal ccRCC Endocrine  Pheochromcytomas CNS  Retinal angiomas  Cerebellar and spinal hemangioblastomas Pancreatic  Pancreatic cysts  Pancreatic neuroendocrine tumors Kidney  Multifocal papillary RCC Skin  Fibrofolliculomas  Trichodiscomas  Achrocordons Lung  Lung cysts Kidney  Oncocytomas  Chromophobe RCC  ccRCC Kidney  Papillary type II RCC

VHL/3p25(111)

HIF regulation Primary cilium Collagen IV homeostasis Fibonectin homesostasis

Bevacizumab (43) Sunitinib (45) Sorafenib (44) Pazopanib (46)

Axitinib Tivozanib

None

XL184 GSK1363089 None

Hereditary papillary RCC (7) Birt Hogg Dube Syndrome (113)

Hereditary leiomyomatosis RCC syndrome (114) Hereditary paraganglioma syndrome Tuberous sclerosis

Endocrine organs  Paragangliomas Kidney  ccRCC CNS  Cortical tubers  Subependymal giant cell astrocytomas (SEGA) Skin  Ash leaf spots  Shagreen patch Kidney  Renal angiomyolipomas  ccRCC

MET/7q31(112) Folliculin/17p11.2(72)

Nutrient Sensing AMPK signaling/ PI3K pathway via mTOR

None

Fumarate hydratase/ 1q42-3(54)

Krebs cycle HIF regulation

None

Bevacizumabþ erlotinib (NCT01130519)

Succinate dehydrogenase D/ 1p36(115)

Krebs cycle

None

None

Hamartin/9q34(116) Tuberin/16p13(117)

PI3 Kinase pathway/mTOR homeostasis

Everolimus for SEGA(118)

Everolimus (multiple studies) Sirolimus Temsirolimus

Preclinical models Rodent knockout models

Clinical genomics of renal tumors

Table 3

Rodent knockout models

Rodent knockout models

Eker Rat Murine knockout models

293

294 clinical evaluation of renal epithelial tumors, even in technically challenging samples, such as FFPE and scant biopsies. Defining the prognosis for a patient with renal cell carcinoma (RCC) is currently more art than science, and is based largely upon algorithms that evaluate clinical features and reflect overall patient health status rather than tumor biology. The recently reported associations of mutation status, gene expression profiles, and chromosomal imbalances with prognosis in ccRCC and pRCC patients suggest that molecular classification of renal epithelial tumors will be an important component of clinical management in these patients. Similarly, despite the fact that therapy for RCC was launched from discoveries in the basic cancer biology of this disease, few reliable biomarkers exist to predict response to targeted therapy. The emergence of targeted therapies directed to specific genetic alterations and the association of specific genetic events with response to therapy would likely reinforce the need for molecular characterization of tumors for therapeutic selection. In addition, novel technologies, such as next-generation sequencing (NGS), might allow for a refined molecular classification of renal tumors, which should inform the development of more targeted agents.

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