Variant Chromophobe A Morphology Not Recognized After Routine Fixation

Ronald J. Cohen, MBBCh, FFPath, PhD, FRCPA; Sydney Weinstein, MBBCh, FRACS; Terry Robertson, PhD; Loryn N. Sellner, PhD; Hugh J. S. Dawkins, PhD; John E. McNeal, MD

● Separation of renal cell tumors into different prognostic (clear cell) renal cell carcinomas, contain abundant mu- groups is an imperative function of the diagnostic pathol- copolysaccharide that reacts positively with Hale’s colloi- ogist. Recently, chromophobe renal carcinoma has been dal iron.2 They lack neutral fat, and although immunore- described as a tumor that is morphologically distinct from active for cytokeratin, they are negative for vimentin stain- conventional ‘‘clear cell’’ carcinoma and that has a low ing. Ultrastructurally, the tumor is characterized by nu- metastatic potential. Identification is based on routine light merous cytoplasmic microvesicles, which are thought to microscopic features and is confirmed by special stains, arise from outpouchings of the cytoplasmic mitochon- immunohistochemistry, and electron microscopy. We pre- dria.6,7 These tumors appear to have a significantly more sent a variant of chromophobe renal carcinoma that did favorable outcome than either conventional clear or gran- not show the typical cytomorphologic features on light mi- ular cell carcinoma.3,4,8 croscopy after formaldehyde fixation. After fixation in Sol- ufix (a commercial fixative), these features were recog- REPORT OF A CASE nized and the diagnosis was confirmed. The tumor also A 71-year-old man presented with recent onset of abdominal showed an unusual form of calcification and psammoma discomfort, and an incidental 4-cm mass was identified on com- body formation not previously recognized in chromophobe puted tomographic scan in the upper pole of his left kidney. The tumors. Molecular biological assessment was inconclusive, patient’s past medical history included a previous diagnosis of but excluded a chromosome 3p deletion usually found in prostatic carcinoma, for which the patient had received external conventional renal carcinoma. The use of a different pri- beam radiation therapy. At this time, there was no evidence of prostatic tumor recurrence, and his serum prostate-specific anti- mary fixative may provide a cost-effective screening tool gen level was 0.1 ng/mL (normal range, Ͻ4 ng/mL). to detect variant renal tumors and may have important Following a left radical nephrectomy, the fresh specimen was prognostic implications. bisected, and a uniform, tan, partly cystic tumor was identified (Arch Pathol Lab Med. 2000;124:904–906) arising from the superficial cortex of the upper pole. The tumor appeared partly encapsulated and did not involve the renal ves- sels. hromophobe renal cell carcinoma is a distinct tumor C type with unique morphologic and cytogenetic fea- MATERIALS AND METHODS tures.1–5 The major histologic feature characterizing these Tumor tissue was fixed in both 4% buffered formaldehyde and carcinomas is their voluminous cell cytoplasm, which has Solufix (Tissue Technologies, Perth, Australia), while frozen un- a pale, finely reticular quality and contrasts with well-de- fixed tissue was sectioned and stained for neutral fat using Sudan fined cell borders. An eosinophilic variant is also recog- IV. Processed tissues (both Solufix and formaldehyde-fixed) were nized, in which tumor cells contain an additional comple- cut at 4 ␮m for routine hematoxylin-eosin, periodic acid–Schiff, ment of mitochondria. Distinction from conventional renal Perls , and von Kossa stains, as well as with stains carcinoma is totally dependent on the identification of for mucopolysaccharides (Hale’s colloidal iron). Immunostains for these subtle features on routinely stained sections, and the vimentin (Dakopatts A/S, Glostrup, Denmark, 1:100) were per- diagnosis is only then confirmed by special stains, electron formed, and cryostat sections were stained for lipid with Sudan microscopy, or both. These tumors, unlike conventional IV. For electron microscopy, thin sections (60–90 nm) were stained with uranyl acetate and lead citrate and were examined in a Phil- lip’s 410LS transmission electron microscope at 80 kV. Radio- graphic microanalysis was also carried out with an energy-dis- Accepted for publication November 5, 1999. persive x-ray analysis detection system (Moran Scientific Soft- From the Departments of Urology, Urological Research Centre (Drs ware, Adelaide, Australia). Cohen, Weinstein, and Dawkins), Pathology (Dr Robertson), and Mo- lecular Pathology (Dr Sellner), University of Western Australia, Perth, To isolate DNA from tumor tissue and normal kidney tissue, 20- Western Australia; and the Department of Urology, Stanford University ␮m paraffin sections were cut and dewaxed in xylene, and then 9 Medical Centre, Stanford, Calif (Dr McNeal). DNA was prepared as previously described. Loss of heterozygosity Dr Cohen has an affiliation with a financial interest in Tissue Tech- was determined by polymerase chain reaction amplification of poly- nologies, a joint venture between the Urological Research Centre and morphic microsatellite markers and gel electrophoresis.10 Markers Uropath. used in this study were against regions previously reported as being Reprints: Ronald J. Cohen, PhD, Urological Research Centre, Level useful in the differential diagnosis of renal cell carcinoma.11 These 2, M Block, The Queen Elizabeth II Medical Centre, Nedlands, Western regions included D1S2883 (chromosome 1), D2S202 (2q32), D3S1675 Australia, 6009, Australia. (3p), D3S1497 (3p22–3p21.3), D3S1514 (3p21–3p14.2), D3S1447 904 Arch Pathol Lab Med—Vol 124, June 2000 Variant Chromophobe Renal Cell Carcinoma—Cohen et al (3p21), D3S1478 (3p21.3–3p21.2), D3S1581 (3p21.2), D6S305 (6q27), D10S1239 (10q23–10q24), D13S317 (13q22), D17S559 (17p13), D17S855 (17q12–17q21), and D21S267 (21q22.1–21q22.3). RESULTS Routine formaldehyde-fixed tissue confirmed a tumor growing in solid sheets, as well as focally having a tubu- loalveolar pattern. Tumor cells had clear bulky cytoplasm, moderate nuclear atypia, and indistinct cell membranes (Figure 1, A). In contrast, tumor tissue fixed in Solufix demonstrated good preservation of cell cytoplasm, which had a fine reticular quality contrasting with sharply de- fined cell borders (Figure 1, B). Dark smudged nuclei seen in the formaldehyde-fixed tissue were not identified in material preserved in Solufix. In contrast, nuclei were clear and demonstrated distinctly clumped chromatin. Because cell membranes were sharply focused, the voluminous cy- toplasm created a characteristic ‘‘plant cell–like’’ appear- ance, a feature not easily recognized in routine formal- dehyde-fixed sections. An unusual feature seen in all sec- tions was the formation of numerous psammoma bodies (Figure 1, C). These bodies seemed to arise from the center of tubuloalveolar structures, which frequently contained eosinophilic material studded with small flecks of calci- um. Histochemical and immunostains were similar in samples fixed in both Solufix and formalin. Stains for Hale’s colloidal iron were strongly positive in the cell cy- toplasm and stained the central amorphous deposits as well as the emerging psammoma bodies. These structures also stained strongly with Prussian blue (Perls) and von Kossa stains, confirming their ferrous and calcium content (Figure 1, C). Periodic acid–Schiff confirmed oc- casional focal positivity for glycogen, and immunostains for vimentin were negative in all tumor cells. Cryostat sec- tions were negative for neutral fat (Sudan IV). Electron Microscopy The tumor consisted of closely apposed polygonal cells, often arranged in tubulelike structures. A prominent basal lamina surrounded each cluster of cells, while small stunt- ed microvilli projected into the narrow intercellular space, as well as into lumenlike spaces (Figure 2). The central proteinaceous material that was noted on light microscopy to form psammoma bodies was recognized as a primarily stromal/basement membrane structure, which contained electron-dense granular material (Figure 2). This material ranged from 25 to 280 nm in diameter and was occasion- ally observed as a fibrillar featherlike structure (Figure 2, Figure 1. A, Formaldehyde-fixed tumor tissue confirms cells with inset). Larger granules were also identified and found on bulky ‘‘clear’’ cytoplasm, irregular nuclei, and indistinct cell borders, energy-dispersive x-ray analysis to contain significant consistent with a low-grade, conventional, clear cell renal carcinoma peaks for iron, calcium, and phosphate, confirming the (hematoxylin-eosin, original magnification ϫ400). B, Tumor tissue fixed light microscopic findings. The main ultrastructural fea- in Solufix confirms rounded nuclei, finely reticular cytoplasm, and dis- tinct cell borders, suggestive of a chromophobe renal carcinoma (he- ture of the tumor cells was the presence of vacuolar and matoxylin-eosin, original magnification ϫ400). C, Stromal accumula- vesicular structures in the cytoplasm, which ranged from tions of mucopolysaccharide speckled with deposits of ferrous salts. 500 to 1600 nm in greatest dimension. They consisted of Inset, Formation of concentrically layered iron-containing psammoma a closed smooth membrane and were round, ovoid, or ir- bodies (formalin-fixed tissue, , original magnification regular in shape. A small proportion contained inner ves- ϫ400). icles measuring 150 to 300 nm in diameter. The vesicular membranes were always smooth, and ribosomes were commonly observed in conventional (clear cell) nonpapil- never seen on their surface. lary renal cell carcinomas. No loss of heterozygosity was observed at D1S2883, D2S202, D6S305, D10S1239, D13S317, Molecular Biological Assessment D17S559, or D21S267. The patient was homozygous at No loss of heterozygosity was observed at D3S1447. The D17S855. Loss of heterozygosity at these loci is also com- patient was homozygous at D3S1675, D3S1497, D3S1514, monly observed in chromophobe renal cell carcinomas and D3S1478. Loss of heterozygosity at chromosome 3p is and occasionally in oncocytomas. Arch Pathol Lab Med—Vol 124, June 2000 Variant Chromophobe Renal Cell Carcinoma—Cohen et al 905 Figure 2. Electron micrograph of core of connective tissue containing electron-dense granular material surrounded by tumor cells (original magnification ϫ4320). True lumen is identified in lower left corner with associated microvilli. The prominent cytoplasmic feature is the presence of vacuolar and vesicular structures. Inset, Confirmation of fibrillar, featherlike, electron-dense granules found within many of these connective tissue cores (original magnification ϫ33 800).

COMMENT cytologic features suggestive of chromophobe renal car- Chromophobe renal carcinoma has a significantly better cinoma were easily appreciated. As formaldehyde is the prognosis than conventional renal carcinoma (clear cell mainstay of tissue fixation, it is highly likely that many type), therefore making it mandatory that distinction be other renal tumor variants have been designated conven- made in each case. Although the gross appearance is often tional clear cell renal cell carcinoma without further in- suggestive, diagnosis rests with its characteristic light mi- vestigation. As the classification of renal carcinoma is in- croscopic appearance. The abundant reticular cytoplasm complete and constantly changing,14 variant tumors such with distinct cell borders often provides the impetus to as this case require identification not only to refine and embark on confirmatory histochemical stains and immu- improve these tumor divisions, but also because of their nostains or, in selected cases, electron microscopy. In this prognostic differences. This patient remains well without case, these light microscopic features were not obvious in any evidence of recurrent disease 12 months after surgery. formaldehyde-fixed tissue and were only noted after fix- Future fixation of at least small samples of tumor in Sol- ation in Solufix. The histochemistry, immunostains, and ufix may avert future misdiagnoses, along with their sub- electron microscopic features of this case all strongly sup- sequent prognostic implications. port the diagnosis of chromophobe carcinoma, and al- References though the molecular markers do not identify any of the 1. Bonsib S, Lager D. carcinoma: analysis of five cases. Am many possible markers of chromophobe carcinoma, they J Surg Pathol. 1990;14:260–267. also do not support the diagnosis of a conventional clear 2. Tickoo S, Amin M, Zarbo R. Colloidal iron staining in renal epithelial neo- cell carcinoma. It is therefore possible that this tumor may plasms, including chromophobe renal cell carcinoma: emphasis on technique and patterns of staining. Am J Surg Pathol. 1998;22:419–424. represent a new variant of renal carcinoma or alternatively 3. Cochand-Priollet B, Molinie V, Bougaran J, et al. Renal chromophobe cell a subtype of chromophobe renal tumors that is as yet un- carcinoma and oncocytoma: a comparative morphologic, histochemical, and im- recognized. Currently, all renal tumors in our laboratory munohistochemical study of 124 cases. Arch Pathol Lab Med. 1997;121:1081– are being assessed in Solufix; we plan to evaluate a wide 1086. 4. Crotty T, Farrow G, Lieber M. Chromophobe cell renal carcinoma: clini- series of tumor types and their clinical outcomes. copathological features of 50 cases. J Urol. 1995;154:964–967. A feature of this tumor is the presence of numerous 5. Billis A, Carvalho R, Magrini E, et al. Chromophobe renal cell carcinoma: psammoma bodies. These bodies are often seen in papil- clinicopathological study of 7 cases. Ultrastruct Pathol. 1998;22:19–26. 12 6. Thoenes W, Storkel S, Rumpelt H-J, et al. Chromophobe cell renal carci- lary renal carcinoma and occasionally in oncocytoma noma and its variants: a report on 32 cases. J Pathol. 1988;155:277–287. (7%).3 The mechanism of psammomatous calcification is 7. Erlandson R, Reuter V. Renal tumor in a 62-year-old male. Ultrastruct Pathol. controversial, and the accepted concept of origin from ne- 1988;12:561–567. 8. Renshaw A, Henske E, Loughlin K, et al. Aggressive variants of chromophobe crotic papillae has been questioned by the presence of hy- renal cell carcinoma. Cancer. 1996;78:1756–1761. droxyapatite, suggesting intracytoplasmic evolution of 9. Turbett GR, Barnett TC, Dillon EK, et al. A single-tube protocol for the ex- psammoma bodies.13 In this case, the origin appears to be traction of DNA or RNA from paraffin-embedded tissues using a starch-based within the stroma adjacent to tumor cells in which accu- adhesive. BioTechniques. 1996;20:846–853. 10. McCulloch R, Sellner L, Papadimitriou J, et al. The incidence of microsat- mulation of mucopolysaccharides, calcium ions, and he- ellite instability and loss of heterozygosity in fibroadenoma of the breast. Breast mosiderin initiate the evolution of the psammoma body. Cancer Res Treat. 1998;49:165–169. The association of psammoma bodies with hemosiderin 11. Bugert P, Kovacs G. Molecular differential diagnosis of renal cell carci- nomas by microsatellite analysis. Am J Pathol. 1996;149:2081–2088. pigment has not been described previously and may be 12. Delahunt B, Eble J. Papillary renal cell carcinoma: a clinicopathologic and an important factor in this unusual form of calcification. immunohistochemical study of 105 tumors. Mod Pathol. 1997;10:537–544. In summary, this case represents a renal tumor that has 13. Murayama H, Kamio A, Imai T, et al. Gastric carcinoma with psammo- the features of a chromophobe carcinoma, but that would matous calcification: report of a case, with reference to calculogenesis. Cancer. 1982;49:788–796. 14 have been classified as conventional clear cell carcinoma 14. Kovacs G, Akhtar M, Beckwith B, et al. The Heidelberg classification of if fixed only in formaldehyde. After fixation in Solufix, renal cell tumours. J Pathol. 1997;183:131–133.

906 Arch Pathol Lab Med—Vol 124, June 2000 Variant Chromophobe Renal Cell Carcinoma—Cohen et al