Chromophobe Renal Cell Carcinoma with Osseous Metaplasia: a Case Report

A 60-year-old Japanese male with a chromophobe cell carcinoma of his left kidney is reported. The tumor, 18 × 27 mm in size, was incidentally found by abdominal ultrasonography. Computed tomography and magnetic resonance imaging demonstrated a well-demarcated solid tumor arising from the lower pole of the left kidney. Histopathological examination of the surgically removed tumor revealed that it was composed of solid sheets of cancer cells having abundant and slightly eosinophilic reticular cytoplasm with accentuated cell membranes making up a plant cell-like appearance. Electron microscopic examination demonstrated numerous intra-cytoplasmic microvesicles. Although the tumor cells were positive for cytokeratin and epithelial membrane antigen, they did not show vimentin immunoreactivity. The unique histological finding of this tumor from other reported renal chromophobe carcinomas was that it had a peripheral fibrotic area with a focus of metaplastic ossification. Key words: renal cell carcinoma – chromophobe cell carcinoma – ossification – immunohistochemistry – electron microscopy


INTRODUCTION
The updated WHO histological classification offers a subdivision of renal cell carcinoma (RCC) by both their morphological and cytogenetic features into clear cell, granular cell, chromophobe cell, spindle cell, cyst-associated, papillary and collecting-duct carcinomas (1).Chromophobe cell carcinoma of the kidney is a recently recognized category of human RCC (2) that was initially described in nitrosomorpholine-induced experimental rat RCC by Bannasch et al. (3).At present, chromophobe RCC may account for about 5% of all cases of RCC.Cancer cells of this distinct type of RCC display a characteristic weak and cloudy cytoplasmic staining pattern imparting a reticular appearance with conventional hematoxylin and eosin stain, corresponding to a variable number of cytoplasmic microvesicles seen by electron microscopy.Genetically, it is characterized by a combination of loss of heterozygosity at chromosomes 1, 2, 6, 10, 13, 17 and 21 (4).Ultrastructural and immunohistochemical characterization have demonstrated the close phenotypic similarity between chromophobe RCC and intercalated cells of the renal collecting duct (5,6).Here we present a case of chromophobe RCC with osseous metaplasia within the tumor tissue.

CLINICAL SUMMARY
A physician incidentally found a tumor of the left kidney in a 60-year-old Japanese male by abdominal ultrasonography in the course of a routine check-up.Computed tomography (CT) and magnetic resonance imaging demonstrated a well-demarcated, solid tumor arising from the lower pole of the left kidney (Fig. 1).The tumor was subsequently removed by partial nephrectomy at the Department of Urology, Hiroshima General Hospital of West Japan Railway Company.
The patient has been followed up for 2 years by abdominal CT taken every 3 months after the surgery without any evidence of tumor recurrence or metastasis.

PATHOLOGICAL FINDINGS
The resected tumor, measuring 18 × 27 mm, was well circumscribed with a fibrous capsule.The cut surface of the tumor was brownish tan with focal hemorrhage.Necrotic area was not observed.Paraffin sections from formalin-fixed material were stained with hematoxylin and eosin, periodic acid-Schiff (PAS), alcian blue (AB) and Hale's colloidal iron.Immunohistochemical examination was undertaken using Vectastain avidin-biotin peroxidase complex kit (Vector, Burlingame, CA) and specific monoclonal antibodies against epithelial membrane antigen (EMA) (Clone GP1.4), cytokeratin (Clone 5D3 and LP34) and vimentin (Clone VIM3B4) as described elsewhere (7).Antibodies were purchased from Novocastra (Newcastle upon Tyne, UK).Histologically, the tumor was composed of solid cell sheets surrounded by delicate stroma containing capillaries (Fig. 2a).The tumor cells had abundant and cloudy cytoplasm imparting a reticular appearance with accentuated cell membranes making up a plant cell-like appearance.The nuclei were slightly pleomorphic with coarsely granular chromatin and occasional prominent nucleoli conforming to a grade 2 nuclear atypia (Fig. 2b).Periodic acid-Schiff (PAS) staining demonstrated a fine and delicate reaction to glycogen in comparison with heavy deposits observed in ordinary clear cell carcinomas (not shown).The cytoplasm of the tumor cells revealed a diffuse positive reaction with Hale's colloidal iron stain (Fig. 2c), which is a diagnostic feature for chromophobe RCC (2).The peripheral part of the tumor underwent sclerotic fibrosis and focal osseous metaplasia was found (Fig. 2d).The tumor cells revealed a positive immunoreaction to EMA and cytokeratin, while vimentin immunoreactivity was exclusively negative throughout the tumor.Electron microscopy of the tumor cell revealed numerous microvesicles within the cytoplasm (not shown).These vesicles, round to oval in shape, were 200-400 nm in diameter and had a tendency to be concentrated adjacent to and surrounding the nucleus.

DISCUSSION
Although limited data are available regarding the clinical behavior of chromophobe RCC, it has been suggested that this distinctive type of RCC has a relatively low malignant potential compared with common type RCC (8,9).Histopathological characteristics of chromophobe RCC are a nesting arrangement of the tumor cells with sharply defined borders (plant-like appearance) and abundant pale acidophilic reticular cytoplasm (2).Since the initial report by Thoenes et al. (2), positive cytoplasmic reaction to Hale's collodal iron stain has been considered to be a diagnostic feature for chromophobe RCC and has been used as a discriminatory feature to differentiate it from other renal tumors.However, it is noted that other subtypes of renal cancer also show positive reaction to this method with different staining patterns in comparison with that described for chromophobe RCCs.Tickoo et al. (10) emphasized that the diffuse and strong reticular cytoplasmic stain with Hale's method, as was observed in tumor cells in the present case, was highly characteristic of chromophobe RCC.The presence of variable numbers of intracytoplasmic microvesicles is an important electron microscopic feature of this type of RCC (2).Bonsib and Lager (9) and Akhtar et al. (11) suggested that these microvesicles may be derived from the outer membranes of mitochondria through extensive ultrastructural study.However, Tickoo et al. (12) could not observe coarse granular cytoplasmic staining by antimitochondrial antibody 113-1, that recognizes a 60 kDa non-glycosylated protein component of mitochondria, in typical chromophobe RCC.Tumor cells of chromophobe RCC present negative vimentin immunoreactivity, while demonstrating positive immunoreaction to cytokeratin and EMA, as was observed with the present tumor (13).This is also one of the characteristic phenotypes of chromophobe RCC for distinction from common-type RCCs.From these morphological observations, we concluded that the present tumor was compatible with typical chromophobe RCC (8).
The unique histopathological finding of the present chromophobe RCC is a focus of bone formation within the tumor.Daniel et al. (14) reported that 10.3% (58/580) of renal cell carcinomas had calcified foci through a roentogenographic review of 2709 renal masses at the Mayo Clinic.Moreover, reports on osseous metaplasia or bone formation within RCC with histopathological confirmation are rare (15,16).Although Akhtar et al. (11) described two cases of chromophobe RCC with radiologically validated calcification, substantially this is the first report on chromophobe RCC with histologically proven ossification.Because the ossified focus in the present case was found within the fibrotic area of the tumor apart from cancer cell nests, stromal osseous metaplasia could be the best explanation for the histogenesis (17).
Recent advances in cytogenetic and molecular biological analysis have disclosed the genetic background of each subtype of RCC (18)

Figure 1 .
Figure 1.Medical imaging of the renal tumor.(a) Coronal images on dynamic magnetic resonance imaging reveal a well-defined, solid tumor arising from the lower pole of the left kidney with inhomogeneous enhancement.(b) Plain computed tomography shows a small calculus within the tumor.
Speicher et al. (4) reported specific loss of chromosomes 1, 2, 6, 10, 13, 17 and 21 in chromophobe RCC by a comparative genomic hybridization method.On the other hand, common-type RCCs are genetically separated from others based on the occurrence of a highly specific deletion of chromosome 3p (19) with mutation of VHL gene (20).Combination of histopathological examination and molecular cytogenetic analysis as was proposed by Bugert and Kovacs (21) may improve the accuracy of diagnosis of RCCs according to the new WHO classification in the near future.

Figure 2 .
Figure 2. Histopathological findings of the tumor resected from the left kidney.(a) The tumor was composed of solid cell sheets with delicate stroma containing capillaries.H&E stain (×27).(b) Tumor cells have abundant reticular cytoplasm with accentuated cell membranes making up a plant cell-like appearance.H&E stain (×131).(c) Cancer cells reveal diffuse positive staining with Hale's colloidal iron stain (×131).(d) Small foci of osseous metaplasia are found at the peripheral sclerotic area of the tumor.H&E stain (×68).