Kidney cancer is among the ten most common causes of cancer-related death in adults 1. Over 64,700 new cases and over 13,500 deaths are expected to occur in the US in 2012 2. Renal cell carcinoma (RCC) constitutes more than 80% of all primary renal neoplasms, and clear cell RCC (ccRCC) accounts for most of these cases (80%). While a specific histological diagnosis is possible by morphology and immunohistochemistry in the majority of cases, overlapping morphological features are still encountered in about 7% of cases in routine practice 3, and these cases fall in the category of "unclassified renal neoplasm." Recent updates to the histopathological diagnosis of kidney neoplasms are reflected in the latest World Health Organization (WHO) classification of genitourinary and kidney neoplasms 4. However, ongoing research has led to the description of new tumor entities that are not yet considered in this classification. One of these recently described entities is tubulocystic renal carcinoma (TCRC) with less than one hundred cases reported 5.

Genetic events play an important role in renal carcinogenesis, and most known renal tumor subtypes have recurrent specific chromosomal imbalances 6. Thus, genetic profiling of renal tumors has emerged as a practical diagnostic tool for the surgical pathologist, especially in those cases where morphologic diagnosis is not conclusive 3. Although the identification of key molecular targets involved in renal carcinogenesis has had direct impact in patient care, little is known about the genetic events involved in tumor progression 6.

Herein we describe the clinico-pathological features of a patient presenting with synchronous ccRCC and a TCRC and the molecular profiles of these tumors utilizing virtual karyotyping. Our results contrast the different profiles of these tumors as well as exemplify the chromosomal imbalances associated with histologic progression from low to highgrade ccRCC.

A 67-year-old previously healthy male presented with acute onset of chest pain. His past medical history was unremarkable except for a history of RCC in his mother. The patient's physical exam and cardiac workup were negative. Two incidental right renal masses were found on CT scan performed as part of his chest pain evaluation (Figure 1). A 3.5 cm, solid, enhancing mass located in the anterior aspect of the upper pole; and a 1.2 cm mass located along the mid lateral aspect of the kidney. The patient denied flank pain, hematuria, or weight loss. Additional workup for metastatic disease was negative. Partial nephrectomy was attempted to remove the lesions; however, a total nephrectomy was ultimately necessary. No adjuvant therapy was administered to the patient. Twenty-four months after the procedure, the patient remains free of disease.

We report a case of two synchronous non-related tumors, a TCRC and a ccRCC occurring in the same kidney with molecular characterization of both neoplasms.

TCRC was originally described by Masson who named it "Bellinian epithelioma" or "carcinoma of the collecting ducts" as he believed it originated from collecting ducts of Bellini 9. MacLennan et al. 10 in 1997 hypothesized that this tumor represented the low grade of the spectrum of collecting duct carcinoma, as it shares similar characteristics with the latter tumor (i.e., immunopositivity for 34βE12 and UEA-1). In 2004, Amin et al. named the tumor "tubulocystic carcinoma" in a series of 29 cases 11.

TCRC is a rare entity that characteristically has a small size at presentation (mean 2 cm) and rarely progresses, recurs, or metastasizes 781213. It has been reported in association with other RCCs, primarily papillary RCC 5. Recent immunohistochemical, ultrastuctural, and molecular studies (RNA expression levels and genomic profile analyses) have shown that TCRC is different from collecting duct carcinoma and may be related to papillary RCCs 578131415. Supportive findings include the expression of proximal convoluted tubule markers (CD10 and AMACR), distal nephron proteins (parvalbumin, HMWCK and CK19) 57, and the detection of cells reminiscent of proximal tubule and intercalated cells by electron microscopy 5131415. Further supporting the notion that TCRC is related to papillary RCC is the report of one group that described that TCRC has a gene expression profile similar to that of papillary RCCs using cluster analysis 8. However, another study showed a distinct gene expression profile characterized by expression of cell cycle and biomolecule metabolism genes although this group did not compare this profile directly to papillary tumors 5. Using fluorescence in situ hybridization (FISH) and array-based comparative genomic hybridization (aCGH), Zhou et al. 13 and Yang et al. 8 have shown gains in chromosome 17, as is seen with papillary RCCs and thus have proposed that these two entities are closely related.

Our case showed classic features of TCRC, such as positive immunohistochemistry for CD10, AMACR and CK19, as well as gains of chromosome 17 in the virtual karyotyping analysis. In addition, the TCRC also showed gains in chromosome 8 and loss of chromosome 9 (Figure 5). The chromosomal imbalance pattern observed in our TCRC case is distinct from that found in papillary RCC which commonly shows gains of 7 and 17 and rarely shows gains of 8 or loss of 9 6. Importantly, loss of chromosome 9 and gain of 17 seem to be recurrent chromosomal imbalances in TCRC tumors (Monzon and Amin, unpublished observations). If this is confirmed in a large case series, it could help establish TCRC as a genetically separate entity from papillary RCC. It is important to note that the cystic and somewhat paucicellular nature of the tumor may be an important consideration when performing molecular analyses of this tumor subtype, since stromal cells may play a role as contaminants. Also, the role of stromal cells in the pathogenesis of TCRC remains uncertain, since metastatic cases not only show the cystic glandular configurations but also the intervening stroma 5.

The differential diagnosis of TCRC includes mixed epithelial and stromal tumor and cystic nephroma 16. The former, another variant of RCC, is easily ruled out as typically displays an ovarian-like stroma that is usually estrogen receptor positive and occurs predominantly in female patients 1718. The so-called cystic nephroma has become basically a non-existent entity 1719. In the WHO classification of renal tumors 4 there is an illustration of a cystic nephroma which looks identical to the TCRC. We suspect that the "cystic nephroma" is actually the tumor now known as TCRC.

There are sporadic reports about the coexistence of TCRC with other renal tumors of different subtypes. Yang et al. reported 5 cases of TCRC associated with either papillary RCC or papillary adenomas 8. Gonul et al. reported a case of synchronous ccRCC, micropapillary urothelial carcinoma and TCRC in a 57-year-old male with hematuria 20. Brennan et al. presented a case of a 72-year-old male with end stage renal disease who developed a TCRC, a type 2 papillary RCC, a clear cell papillary and cystic RCC as well as renal oncocytosis, hybrid tumors and chromophobe RCC 21. More recently, Deshmukh et al. reported a synchronous TCRC and papillary RCC in a young female with metastatic papillary RCC in para-aortic lymph nodes 22. Thus, although TCRC has been reported in association with multiple other renal cell tumor subtypes, it appears that there is a slight predominance for synchronous TCRC and papillary tumors. An interesting question raised by the coexistence of TCRC with other renal tumors is whether there are common predisposing factors for these histologically different tumors.

The ccRCC in this case showed a spectrum of morphologic variations ranging from low grade to high grade and a distinct nodule with oncocytic changes which allowed us to inquire about molecular differences within morphologic progression in a single tumor. We analyzed the areas in the ccRCC showing different grade and morphology using virtual karyotyping and demonstrated that a higher Fuhrman grade correlated with an increase in chromosomal imbalances (Figure 5). In a similar fashion, studies on chromophobe RCCs have shown that multiple chromosomal gains are associated with sarcomatoid transformation 23. More recently, Patani et al. using high-resolution aCGH and FISH have also demonstrated that morphologically distinct regions from a case of triple-negative breast carcinoma (apocrine vs. non-apocrine areas) correlate with different genetic aberrations 24.

In addition to the somatically acquired genetic changes in the tumors, the patient's tumor had regions of inherited uniparental disomy (UPD, i.e. two chromosomal regions inherited from one parent only) in chromosomes 2 and 6 (Figure 5, asterisks). The presence of these genetic lesions in the normal kidney tissue suggests a possible inherited origin. Although uniparental disomy has been implicated as a risk factor for colon and other cancers, its role in the predisposition of renal tumors is currently unknown 25.

In conclusion, this case allowed us to explore two uses of whole genome analysis with virtual karyotyping. The first was to confirm that ccRCC and TCRC have distinct genomic copy number profiles and that TCRC appears to have recurrent gains in chromosome 17 which might be implicated in the pathogenesis of this tumor. Second, the presence of a ccRCC with varying degrees of differentiation allowed us to evaluate the sequence of chromosomal imbalances acquired during tumor progression. These findings support the concept that chromosomal imbalances are associated with tumor progression 26, and possibly explain why Fuhrman grading is a strong prognostic factor in ccRCC 27 even when high grade cells are present in a minority of the specimen 28.

This study was conducted under a local IRB approved protocol for the molecular characterization of renal tumors.

The authors declare that they have no competing interests.

GQG and SPO retrieved clinical information and wrote the manuscript. RG, GQG, KCO, MRS and AA first identified this case, proposed the studies and provided valuable insight during manuscript preparation. FAM provided the molecular analysis data and supervised manuscript preparation. All authors reviewed and approved the final manuscript.