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Renal Oncocytoma: Clinical and Biological Correlates
0022-534 7/93/1505-1380$03.00/0
Vol. 150, 1380-1383, November 1993 Printed in U. S.A.
THE JOURNAL OF UROLOGY Copyright © 1993 by AMERICAN UROLOGICAL ASSOCIATI ON , INC.
RENAL ONCOCYTOMA: CLINICAL AND BIOLOGICAL CORRELATES MARK R. LICHT, ANDREW C. NOVICK,* RAYMOND R. TUBBS, ERIC A. KLEIN, HOWARD S. LEVIN AND STEVAN B. STREEM From the Departments of Urology and Pathology, The Cleveland Clinic Foundation, Cleveland, Ohio
ABSTRACT
We treated 31 patients with renal oncocytoma. Renal cell carcinoma was found existing separately within the same or contralateral kidney in 10 patients (32% ). Followup of 29 patients revealed 24 alive with no evidence of disease and 1 alive with recurrent oncocytoma, while 1 with coexistent tumors died of progressive renal cell carcinoma and 3 tumor-free patients died of unrelated diseases. Quantitative deoxyribonucleic acid analysis was performed on cell suspensions of fresh tumor by flow cytometry or by image cytometry on touch preparations from frozen tissue in 16 patients with renal oncocytoma. Ploidy analysis revealed all oncocytomes to be diploid. Frozen tissue immuno histology was performed using murine monoclonal antibody against human HLA-A, B and C (class I) antigens with the avidin-biotin peroxidase technique in 11 patients with renal oncocytoma. Ten oncocytomas did not express these self-recognition antigens and 1 was only weakly positive for antigen expression. In contrast, renal cell carcinomas strongly expressed HLA class I antigens. The high incidence of coexistence of renal oncocytoma and renal cell carcinoma has important clinical implications. Loss of HLA class I antigen expression by renal oncocytomas may provide an additional method for differentiating this lesion from renal cell carcinoma. KEY WORDS: kidney neoplasms, oncocytoma Renal oncocytoma was first characterized histologically by Hamperl,1 · 2 and later described by Klein and Valensi. 3 The benign nature of this tumor has been documented by many investigators. 4-7 The recent widespread use of abdominal ultra sound and computerized tomography (CT) has increased the number of incidentally detected solid renal masses. 8 • 9 However, renal oncocytoma is most often indistinguishable from renal cell carcinoma by common diagnostic imaging modalities. Renal oncocytoma also shares some histological features with renal cell carcinoma, which can make the establishment of a final pathological diagnosis difficult. Investigators have at tempted to distinguish between these 2 tumors by immuno histochemical staining to determine cellular origin, 1 0-12 by analysis of nuclear deoxyribonucleic acid (DNA) ploidy13-15 and by demonstration of chromosomal patterns unique to oncocy tomas. 16· 17 HLA-A, B, C or class I antigens are the principal self recognition antigens in cell-mediated cytolysis. These antigens are expressed on the cell membrane of nucleated cells, including normal renal tubular cells. 1 8 Loss of HLA class I antigen expression has been found in some urological' 9• 20 and non urological21· 22 tumors. However, renal cell carcinoma has been shown to express HLA class I antigens. 2 3 · 24 We review our clinical experience with renal oncocytoma to determine the incidence of coexistence with renal cell carcinoma and also to analyze the HLA class I antigen expression status of oncocy tomas as a potential method for differentiating between these 2 tumors. MATERIALS AND METHODS
Clinical and pathological. Between January 1981 and Decem ber 1991, 31 patients with the pathological diagnosis of renal oncocytoma were identified at our clinic. The diagnosis was made on review of microscopic sections of either partial or radical nephrectomy specimens. All tumors were evaluated by 1 pathologist (H. S. L.), and strictly fit the diagnostic criteria for renal oncocytoma established by Klein and Valensi3 as
modified by Davis et al. 25 Oncocytomas were characterized as discrete, circumscribed, usually brown masses, often with a central stellate scar. Microscopically, the tumors were com posed of solid nests, cysts or tubular aggregates of cells with granular eosinophilic cytoplasm and usually with uniform nu clei similar in size to the cells of the proximal convoluted tubules of adjacent kidney. Numerous cells of oncocytoma contain nucleoli. Thus, the presence of nucleoli was of no use in grading oncocytoma or separating oncocytoma from renal cell carcinoma. Although nuclear atypia and pleomorphism were originally criteria for excluding the diagnosis of oncocy toma, it has subsequently been shown by Davis et al that tumors with gross and microscopic features of oncocytoma behave like oncocytomas even if they have nuclear atypia but lack mitotic activity and other features of granular type renal cell carci noma, including marked vascularity, focal intercytoplasmic clearing and "pavement-like or tubular growth pattern with sharp cell borders and intercellular cohesion." 25 DNA ploidy analysis. DNA ploidy analysis was performed by either flow or image cytometry of ethanol-fixed whole cells. Fresh renal oncocytoma tumor tissue to be analyzed by flow cytometry was prepared by combined mechanical and enzy matic degradation, and cellular viability was assessed by trypan blue exclusion. Approximately 1 million cells were then washed, incubated and stained with propidium iodine staining solution. Between 15,000 and 30,000 cells from each sample were ana lyzed using the FACScant flow cytometer. Ethanol-fixed hu man lymphocytes served as controls. Touch preparations of frozen tumor tissue were used for image cytometry. Touch preparations were fixed and Feulgen stained. The preparation was then placed in 5 N. hydrochloric acid to hydrolyze the DNA. The slides were then dyed, rinsed, dehydrated and coverslipped. DNA was then measured from 100 to 200 cells of each tumor with the CAS 100+ in a standard fashion, which has been described previously. 26 Histograms containing near-tetraploid populations were classified as dem onstrating near-tetraploid DNA aneuploidy if the G2+M frac tion exceeded 8% and if there was evidence of cycling activity
Accepted for publication April 8, 1993. * Requests for reprints: Department of Urology, The Cleveland t Becton Dickinson, Mountain View, California. Clinic Foundation, 1 Clinic Center Drive, Cleveland, Ohio 44195. :j: Cell Analysis Systems, Lombard, Illinois. 1380
RENAL ONCOCYTOMA
of the near-tetraploid population in a dot plot displaying FL2 versus area. HLA class I antigen exp ression. This immunohistological staining process has been described in detail previously. 27 Fro zen 6 µ. tumor tissue sections from renal oncocytoma were prepared and allowed to air dry on poly-L-lysine-coated slides. The tissue was first subjected to an avidin-biotin flush to minimize nonspecific binding to endogenous biotin and then mouse monoclonal antibody against human HLA-A, B, C an tigens was applied to the sections. Biotinylated anti-mouse IgG and the Elite ABC reagent (avidin-biotinylated peroxidase complex, Vector, No. PK-6 1 00) was then applied to the tissue and the color reaction product was developed with 3-amino-9ethylcarbazole in the presence of 0.005 % hydrogen peroxide. The tissue was then counterstained with fresh hematoxylin and the slides were mounted. The slides were read by 1 experienced immunopathologist (R. R. T.) to determine the presence or absence of HLA-A, B, C antigen expression on the tumor cells. RESULTS
Clinical findings and outcome. The 31 patients in this series
include 22 men and 9 women 40 to 84 years old (mean 63 years) . In 21 patients a renal mass was incidentally discovered during evaluation for a nonurological disorder. Five patients presented with hematuria, 4 with flank pain and 1 with a palpable abdominal mass. A CT scan was performed in 29 patients and none revealed a central stellate scar. Renal arte riography was obtained in 27 patients and only 1 of these studies demonstrated the spoke-wheel vascular pattern de scribed for renal oncocytoma. In all 31 patients renal cell carcinoma was the preoperative diagnosis. Of 24 patients with a unilateral renal mass 19 underwent radical nephrectomy, while 5 were treated with a nephron sparing operation. Of the 7 patients who presented with bilat eral renal masses 5 underwent bilateral partial nephrectomy and 2 underwent partial nephrectomy followed by radical ne phrectomy of the contralateral kidney. One patient underwent a second partial nephrectomy for a locally recurrent tumor 5 years postoperatively. A final pathological diagnosis of oncocytoma was established in all 31 patients. Oncocytoma was present unilaterally in 27 patients and bilaterally in 4. Multiple oncocytomas were found in 5 of 35 affected kidneys. The oncocytomas varied in size from 1 to 13 cm. (mean 3. 7 ) . Focal hemorrhage was occasionally observed within an oncocytoma but none of the tumors dem onstrated capsular penetration or venous invasion. Renal cell carcinoma and renal oncocytoma were found in 10 patients (32 % ) . In 7 patients both tumors were present as separate lesions within the same kidney. In 3 patients renal cell carcinoma and oncocytoma were found in opposite kidneys.
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One of the latter patients, initially treated with bilateral partial nephrectomy, had a local tumor recurrence 5 years later in the kidney that had contained renal cell carcinoma. Repeat partial nephrectomy of this kidney revealed the recurrent tumor to be an oncocytoma. Among patients with renal cell carcinoma and oncocytoma, the renal cell carcinoma displayed a granular cell histology in 5, a clear cell histology in 4, and a mixed granular and clear cell histology in 1. One patient was originally diag nosed in 1984 as having renal cell carcinoma developing within a circumscribed oncocytoma. Upon review of the sections in preparation for this article, it was recognized that the foci of marked cellular enlargement, multinucleation and nuclear atypia were as described by Davis et al in 1991 and were characteristic of their cases of atypical oncocytomas with cell ular pleomorphism. 25 After review, this oncocytoma was not considered to have an associated renal cell carcinoma. All other renal cell carcinomas were histologically confirmed to be ma lignancies. Followup was available for 29 patients and ranged from 2 to 122 months (mean 45.6). Of the patients 24 are alive with no evidence of disease, 1 is alive and asymptomatic with residual oncocytoma, 1 with coexistent tumors died of progressive renal cell carcinoma and 3 tumor-free patients died of unrelated diseases. No patient had metastases from oncocytoma. DNA ploidy analysis. Tissue samples from 16 oncocytomas were analyzed for DNA ploidy. In 5 patients the oncocytoma tissue was analyzed by flow cytometry and in 7 it was analyzed by image cytometry. Both methods were used in 4 patients. In all cases the oncocytomas had diploid DNA histograms. HLA class I antigen expression. HLA-A , B, C (class I) antigen expression was determined in 11 renal oncocytomas. Ten on cocytomas showed no HLA-A, B, C antigen expression (see figure) , while 1 showed only trace evidence of expression of these antigens. DISCUS S I O N
Renal oncocytoma is a benign tumor of renal tubular origin. As originally defined by Klein and Valensi, it is a lesion composed of solid sheets of large polygonal eosinophilic cells usually with no cellular anaplasia. 3 Leiber et al described higher grade oncocytomas with the ability to metastasize and cause patient death. 28 However, in their study no patient with a grade 1 oncocytoma experienced metastases or died of the tumor. Davis et al recently suggested that nuclear atypia and cellular pleomorphism are not necessarily evidence for aggressive be havior of an oncocytoma, 25 and we have documented 1 such case as well. Our patient outcome data support the benign nature of this tumor with no instances of metastases and a 100% tumor-specific survival rate at a mean followup of 45.6 months.
A, renal oncocytoma cells show no HLA class 1 antigen expression by immunohistochemical staining. HLA class 1 antigen expression is demonstrated by vascular endothelial cells and macrophages. Reduced from X400. B, renal cell carcinoma demonstrates strong HLA class 1 antigen expression as evidenced by red-orange immunohistochemical reaction product uniformly covering tumor cell membranes. Reduced from X l60.
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1382
RENAL ONCOCYTOMA
On review of the literature, renal oncocytoma is discovered as an incidental finding in approximately 66% of the cases. 29 In our study renal oncocytoma was found incidentally in 21 of 3 1 patients ( 68 % ) during evaluation for a nonurological disor der. Multifocal and bilateral renal oncocytomas were observed in 5 ( 1 6 % ) and 4 ( 1 3 % ) patients, respectively. Renal oncocy toma and renal cell carcinoma have infrequently been found as coexisting lesions within the same 30 or opposite 31 kidney. In a recent review of 1 6 6 patients with oncocytoma, renal cell car cinoma was also present in 7 . 2 % of the patients; in 3 renal cell carcinoma was observed as a small focus within an oncocy toma. 2 5 In our study we found coexisting oncocytoma and renal cell carcinoma in 10 of 31 patients ( 3 2 % ). While this high incidence of coexistence of renal cell carcinoma and oncocy toma may partly reflect the unusual nature of our patient referral population, we believe that this is an important finding with significant clinical implications. It is presently impossible to establish a preoperative diagnosis of renal oncocytoma through radiographic imaging studies. The CT finding of a central stellate scar32 or a spoke-wheel appear ance on angiography33 is not specific and can be seen with renal cell carcinoma. In this study no oncocytoma exhibited a central stellate scar on CT and only 1 tumor had a spoke-wheel pattern on angiography. Some investigators have recommended fine needle aspiration for evaluation of the indeterminant solid renal mass, suggesting that if the cytological findings are con sistent with oncocytoma then conservative surgery3 4 or possibly observation may be warranted. Even if it were possible to establish a prospective diagnosis of oncocytoma, the large num ber of patients in our study with concomitant renal cell carci noma in the same kidney argues against this approach unless a specific indicatio n for nephron sparing surgery is present. 3 5 Many investigators have attempted to differentiate renal oncocytoma and renal cell carcinoma on a cellular and molec ular level. Immunohistochemical staining to determine the tubular cell of origin 1 0- 1 2 and flow cytometry analysis of tumor cell DNA ploidy 1 3 · 1 4 have yielded mixed results. In our study all 16 oncocytomas sampled were diploid, while Rainwater et al found aneuploid DNA in 11 % of grade 1 oncocytomas. 13 Unique chromosomal rearrangement has been found in renal oncocytomas 3 6 and oncocytomas do not display the loss of alleles at loci on the s hort arm of chromosome 3 that is characteristic of renal cell carcinoma. 1 7 Using the anti-renal cell carcinoma monoclonal antibody 5F4, Kochevar and Coffield also showed that higher grade oncocytoma cells stained positive for this renal cell carcinoma specific antigen while grade 1 oncocytoma cells did not. 3 7 We studied the expres sion of HLA-A, B , C or class I antigens on renal oncocytomas as a possible method of differentiating this lesion from renal cell carcinoma. HLA class I antigens are cell membrane -bound proteins that serve as the principal self recognition antigen s in cell-mediated immunity. They are pres ent on almost all nucleated cells and have been shown to be present on normal renal tubular cells. 1 8 Natali et al showed intact HLA class I antigen expression in 9 renal cell carcinomas with no mention of tumor grade, 23 while Kinjo et al demon strated similar antigen expression in 1 1 grade 1 and 2 tumors. 2 4 Data from our laboratory using immunohistochemical staining with monoclonal antibody by the avidin-biotin technique have shown expression of HLA class I antigens by 67 of 68 grades 1 to 3 renal cell carcinomas. 38 Our present results using this same technique in 11 patients demonstrated that 10 oncocytomas did not express HLA class I antigens, while 1 showed only minimal antigen expression. In some tumors cellular loss of HLA class I antigen expres sion has been associated with the presence of malignancy1 9-22 or with progressive tumor dedifferentiation. 2 1 The paradoxical maintenance of HLA class I antigen expression in renal cell carcinoma and loss of such expression in benign renal oncocy tomas are not readily explained. Nevertheless, if these obser-
vations are validated by further experimentation, they may offer an additional method for differentiating oncocytoma from renal cell carcinoma and thereby help to predict tumor behav10r. REFERENCES 1. Hamper!, H.: Benign and malignant oncocytoma.Cancer, 15: 1019, 1962. 2. Hamper!, H.: Oncocytes and oncocytoma. Virch.Arch.Path.Anat., 335: 452, 1962. 3. Klein, M. J. and Valensi, Q. J.: Proximal tubular adenomas of the kidney with so-called oncocytic features. A clinicopathologic study of 13 cases of a rarely reported neoplasm.Cancer, 38: 906, 1976. 4. Harrison, R. H., Baird, J. M. and Kowierschke, S. W.: Renal oncocytoma: ten-year follow-up. Urology, 17: 596, 1981. 5. Merino, M. J. and Livolsi, V. A.: Oncocytomas of the kidney. Cancer, 50: 1852, 1982. 6. Maatman, T. J., Novick,A. C., Tancinco, B. F., Vesoulis, Z., Levin, H. S., Montie, J. E. and Montague, D. K.: Renal oncocytoma: a diagnostic and therapeutic dilemma. J. Urol., 132: 878, 1984. 7. Spring, D. B., Ulirsch, R. C., Starke, W. R. and Brown, S., Jr.: Renal oncocytoma followed for eighteen years without resection. Urology, 26: 389, 1985. 8. Konnak, J. W. and Grossman, H. B.: Renal cell carcinoma as incidental finding. J. Urol., 134: 1094, 1985. 9. Thompson, I. M. andPeek, M.: Improvement in survival of patients with renal cell carcinoma-the role of the serendipitously de tected tumor. J. Urol., 140: 487, 1988. 10. van Krieken, J. H. J. M., Oosterwijk, E., Lycklama a Nijeholt, A. A. B. and Ruiter, D. J.: Renal oncocytoma: a report of two cases with immunohistochemical evaluation. Hum. Path., 19: 460, 1988. 11. Cohen, C., McCue, P. A. and Derose, P. B.: Histogenesis of renal cell carcinoma and renal oncocytoma. An immunohistochemical study. Cancer, 62: 1946, 1988. 12. Eble, J. N. and Hull, M. T.: Glycoconjugate expression in human renal oncocytomas. A lectin histochemical study. Arch. Path. Lab. Med., 112: 805, 1988. 13. Rainwater, L. M., Farrow, G. M. and Lieber, M. M.: Flow cytometry of renal oncocytoma: common occurrence of deoxyribonucleic acid polyploidy and aneuploidy. J. Urol., 135: 1167, 1986. 14. Eble, J. N. and Sledge, G.: Cellular deoxyribonucleic acid content of renal oncocytomas: flow cytometric analysis of paraffin-em bedded tissues from eight tumors. J. Urol., 136: 522, 1986. 15. Hartwick, R. W. J., El-Naggar, A. K., Ro, J. Y., Srigley, J. R., McLemore, D. D., Jones, E. C., Grignon, D. J., Thomas, M. J. and Ayala, A. G.: Renal oncocytoma and granular renal cell carcinoma. A comparative clinicopathologic and DNA flow cy tometric study. Amer. J. Clin. Path., 98: 587, 1992. 16. Kovacs, G., Welter, C., Wilkens, L., Elin, N. and Deriese, W.: Renal oncocytoma. A phenotypic and genotypic entity of renal parenchymal tumors. Amer. J. Path., 134: 967, 1989. 17. Brauch, H., Tory, K., Linehan, W. M., Weaver, D. J., Lovell, M. A. and Zbar, B.: Molecular analysis of the short arm of chro mosome 3 in 5 renal oncocytomas. J. Urol., 143: 622, 1990. 18. Hart, D. N. J., Fuggle, S. V., Williams, K. A., Fabre, J. W., Ting, A. and Morris,P. J.: Localization of HLA-ABC and DR antigens in human kidney. Transplantation, 31: 428, 1981. 19. Nouri, A. M. E., Smith, M. E. F., Crosby, D. and Oliver, R. T. D.: Selective and non-selective loss of immunoregulatory molecules (HLA-A,B,C antigens and LFA-3) in transitional cell carcinoma. Brit. J. Cancer, 62: 603, 1990. 20. Ottesen, S. S., Skouv, J. and Kieler, J.: Reduced HLA-A,B,C expression in tumourigenic v-raf transfected human urothelial cells. Eur. J. Cancer, 26: 305, 1990. 21. Moller, P., Herrmann, B., Moldenhauer, G. and Momburg, F.: Defective expression of MHC class I antigens is frequent in B eel! lymphomas of high-grade malignancy. Int. J. Cancer, 40: 32, 1987. 22. Momburg, F., Ziegler, A., Harpprecht, J., Moller,P., Moldenhauer, G. and Hiimmerling, G. J.: Selective loss of HLA-A or HLA-B antigen expression in colon carcinoma. J. Immunol., 142: 352, 1989. 23. Natali, P. G., Bigotti, A., Nicotra, M. R., Viora, M., Manfredi, D. and Ferrone, S.: Distribution of human class I (HLA-A,B,C)
RE l\,U\.L G :N C O CYTOMA
24. 25. 26.
27. 28. 29. 30. 31.
histocompatibility antigen s in nonnal and rnct11;;,c;m1c tissues cf nonlymphoid origin. Cancer Res . , 44: 4679, Kinjo, T., Takashi, M., Miyake, K. and Nagura, H.: In situ dem onstration of immunological heterogeneity in vascular endothe lial cells of renal cell carcinoma. J. Surg. Oncol., 4 3 : 73, 1990. Davis, C . J., Jr., Mostofi, F. K., Sesterhenn, I. A. and Ho, C . K.: Renal oncocytoma. Clinicopathologica! study of 166 patients. J. Urogen. Path., l: 42, 199 1 . Bauer, T. W . , Tubbs, R . R . , Edinger, M. G . , Suit, P. F . , Gephardt, G. N. and Levin, H. S . : A prospective comparison of DNA quantitation by image and flow cytometry. Amer. J. Clin. Path., 9 3 : 322, 1990. Tubbs, R . R., Sheibani, K., Deodhar, S. D. and Hawk, W . A.: Enzyme immunohistochemistry: review of technical aspects and diagnostic applications. Cleve. Clin. Quart., 48: 245, 1981. Lieber, M . M., Tomera, K. M . and Farrow, G. M.: Renal oncocy toma. J. Urol., 1 2 5 : 481, 1981. Lieber, M . M., Hosaka, Y. and Tsukamoto, T.: Renal oncocytoma. World J. Urol. , 5: 7 1 , 1987. Lehmann, H. D. and Blessing, M. H.: Renal oncocytoma (pathol ogy, preoperative diagnosis, therapy) . Prog. Clin. Biol. Res., 1 00 : 589, 1982. Kavoussi, L. R., Torrence, R . J. and Catalona, W. J.: Renal onco-
32.
33. 34. 35.
36. 37. 38.
]383
with synchronous contralateral renal cell carcinon1a. J. 134: 1 193, 1985. Quinn, M. J., Hartman, D. S., Friedman, A . C., Sherman, J. L., Lautin, E. M., Pyatt, R. S., Ho, C. K., Csere, R. and Fromowitz, F. B . : Renal oncocytoma: new observations. Radiology, 1 5 3 : 49, 1984. Ambos, M. A., Bosniak, M. A., Valensi, Q. J . , Madayag, M. A. and Lefleur, R. S . : Angiographic patterns in renal oncocytomas. Radiology, 1 2 9 : 615, 1978. Cochand-Priollet, B . , Rothschild, E., Chagnon, S . , Nezelof, C . , Debure, A. a n d Galian, A.: Renal oncocytoma diagnosed b y fine needle aspiration cytology. Brit. J. Urol., 6 1 : 534, 1988. Novick, A. C., Streem, S., Montie, J. E., Pontes, J . E., Siegel, S . , Montague, D. K. a n d Goormastic, M.: Conservative surgery for renal cell carcinoma: a single-center experience with 100 pa tients. J. Urol., 1 4 1 : 835, 1989. Walter, T. A., Pennington, R. D., Decker, H. -J. H. and Sandberg, A. A.: Translocation t(9;1 1) (p23;q12): a primary chromosomal change in renal oncocytoma. J. Urol., 1 4 2 : 1 1 7, 1989. Kochevar, J. and Coffield, K. S.: Differential immunostaining of oncocytic renal tumors with an anti-renal cell carcinoma mono clonal antibody. J. Urol., 1 4 1 : 625, 1989. Tubbs, R. R.: Unpublished data.
Vol. 150, 1380-1383, November 1993 Printed in U. S.A.
THE JOURNAL OF UROLOGY Copyright © 1993 by AMERICAN UROLOGICAL ASSOCIATI ON , INC.
RENAL ONCOCYTOMA: CLINICAL AND BIOLOGICAL CORRELATES MARK R. LICHT, ANDREW C. NOVICK,* RAYMOND R. TUBBS, ERIC A. KLEIN, HOWARD S. LEVIN AND STEVAN B. STREEM From the Departments of Urology and Pathology, The Cleveland Clinic Foundation, Cleveland, Ohio
ABSTRACT
We treated 31 patients with renal oncocytoma. Renal cell carcinoma was found existing separately within the same or contralateral kidney in 10 patients (32% ). Followup of 29 patients revealed 24 alive with no evidence of disease and 1 alive with recurrent oncocytoma, while 1 with coexistent tumors died of progressive renal cell carcinoma and 3 tumor-free patients died of unrelated diseases. Quantitative deoxyribonucleic acid analysis was performed on cell suspensions of fresh tumor by flow cytometry or by image cytometry on touch preparations from frozen tissue in 16 patients with renal oncocytoma. Ploidy analysis revealed all oncocytomes to be diploid. Frozen tissue immuno histology was performed using murine monoclonal antibody against human HLA-A, B and C (class I) antigens with the avidin-biotin peroxidase technique in 11 patients with renal oncocytoma. Ten oncocytomas did not express these self-recognition antigens and 1 was only weakly positive for antigen expression. In contrast, renal cell carcinomas strongly expressed HLA class I antigens. The high incidence of coexistence of renal oncocytoma and renal cell carcinoma has important clinical implications. Loss of HLA class I antigen expression by renal oncocytomas may provide an additional method for differentiating this lesion from renal cell carcinoma. KEY WORDS: kidney neoplasms, oncocytoma Renal oncocytoma was first characterized histologically by Hamperl,1 · 2 and later described by Klein and Valensi. 3 The benign nature of this tumor has been documented by many investigators. 4-7 The recent widespread use of abdominal ultra sound and computerized tomography (CT) has increased the number of incidentally detected solid renal masses. 8 • 9 However, renal oncocytoma is most often indistinguishable from renal cell carcinoma by common diagnostic imaging modalities. Renal oncocytoma also shares some histological features with renal cell carcinoma, which can make the establishment of a final pathological diagnosis difficult. Investigators have at tempted to distinguish between these 2 tumors by immuno histochemical staining to determine cellular origin, 1 0-12 by analysis of nuclear deoxyribonucleic acid (DNA) ploidy13-15 and by demonstration of chromosomal patterns unique to oncocy tomas. 16· 17 HLA-A, B, C or class I antigens are the principal self recognition antigens in cell-mediated cytolysis. These antigens are expressed on the cell membrane of nucleated cells, including normal renal tubular cells. 1 8 Loss of HLA class I antigen expression has been found in some urological' 9• 20 and non urological21· 22 tumors. However, renal cell carcinoma has been shown to express HLA class I antigens. 2 3 · 24 We review our clinical experience with renal oncocytoma to determine the incidence of coexistence with renal cell carcinoma and also to analyze the HLA class I antigen expression status of oncocy tomas as a potential method for differentiating between these 2 tumors. MATERIALS AND METHODS
Clinical and pathological. Between January 1981 and Decem ber 1991, 31 patients with the pathological diagnosis of renal oncocytoma were identified at our clinic. The diagnosis was made on review of microscopic sections of either partial or radical nephrectomy specimens. All tumors were evaluated by 1 pathologist (H. S. L.), and strictly fit the diagnostic criteria for renal oncocytoma established by Klein and Valensi3 as
modified by Davis et al. 25 Oncocytomas were characterized as discrete, circumscribed, usually brown masses, often with a central stellate scar. Microscopically, the tumors were com posed of solid nests, cysts or tubular aggregates of cells with granular eosinophilic cytoplasm and usually with uniform nu clei similar in size to the cells of the proximal convoluted tubules of adjacent kidney. Numerous cells of oncocytoma contain nucleoli. Thus, the presence of nucleoli was of no use in grading oncocytoma or separating oncocytoma from renal cell carcinoma. Although nuclear atypia and pleomorphism were originally criteria for excluding the diagnosis of oncocy toma, it has subsequently been shown by Davis et al that tumors with gross and microscopic features of oncocytoma behave like oncocytomas even if they have nuclear atypia but lack mitotic activity and other features of granular type renal cell carci noma, including marked vascularity, focal intercytoplasmic clearing and "pavement-like or tubular growth pattern with sharp cell borders and intercellular cohesion." 25 DNA ploidy analysis. DNA ploidy analysis was performed by either flow or image cytometry of ethanol-fixed whole cells. Fresh renal oncocytoma tumor tissue to be analyzed by flow cytometry was prepared by combined mechanical and enzy matic degradation, and cellular viability was assessed by trypan blue exclusion. Approximately 1 million cells were then washed, incubated and stained with propidium iodine staining solution. Between 15,000 and 30,000 cells from each sample were ana lyzed using the FACScant flow cytometer. Ethanol-fixed hu man lymphocytes served as controls. Touch preparations of frozen tumor tissue were used for image cytometry. Touch preparations were fixed and Feulgen stained. The preparation was then placed in 5 N. hydrochloric acid to hydrolyze the DNA. The slides were then dyed, rinsed, dehydrated and coverslipped. DNA was then measured from 100 to 200 cells of each tumor with the CAS 100+ in a standard fashion, which has been described previously. 26 Histograms containing near-tetraploid populations were classified as dem onstrating near-tetraploid DNA aneuploidy if the G2+M frac tion exceeded 8% and if there was evidence of cycling activity
Accepted for publication April 8, 1993. * Requests for reprints: Department of Urology, The Cleveland t Becton Dickinson, Mountain View, California. Clinic Foundation, 1 Clinic Center Drive, Cleveland, Ohio 44195. :j: Cell Analysis Systems, Lombard, Illinois. 1380
RENAL ONCOCYTOMA
of the near-tetraploid population in a dot plot displaying FL2 versus area. HLA class I antigen exp ression. This immunohistological staining process has been described in detail previously. 27 Fro zen 6 µ. tumor tissue sections from renal oncocytoma were prepared and allowed to air dry on poly-L-lysine-coated slides. The tissue was first subjected to an avidin-biotin flush to minimize nonspecific binding to endogenous biotin and then mouse monoclonal antibody against human HLA-A, B, C an tigens was applied to the sections. Biotinylated anti-mouse IgG and the Elite ABC reagent (avidin-biotinylated peroxidase complex, Vector, No. PK-6 1 00) was then applied to the tissue and the color reaction product was developed with 3-amino-9ethylcarbazole in the presence of 0.005 % hydrogen peroxide. The tissue was then counterstained with fresh hematoxylin and the slides were mounted. The slides were read by 1 experienced immunopathologist (R. R. T.) to determine the presence or absence of HLA-A, B, C antigen expression on the tumor cells. RESULTS
Clinical findings and outcome. The 31 patients in this series
include 22 men and 9 women 40 to 84 years old (mean 63 years) . In 21 patients a renal mass was incidentally discovered during evaluation for a nonurological disorder. Five patients presented with hematuria, 4 with flank pain and 1 with a palpable abdominal mass. A CT scan was performed in 29 patients and none revealed a central stellate scar. Renal arte riography was obtained in 27 patients and only 1 of these studies demonstrated the spoke-wheel vascular pattern de scribed for renal oncocytoma. In all 31 patients renal cell carcinoma was the preoperative diagnosis. Of 24 patients with a unilateral renal mass 19 underwent radical nephrectomy, while 5 were treated with a nephron sparing operation. Of the 7 patients who presented with bilat eral renal masses 5 underwent bilateral partial nephrectomy and 2 underwent partial nephrectomy followed by radical ne phrectomy of the contralateral kidney. One patient underwent a second partial nephrectomy for a locally recurrent tumor 5 years postoperatively. A final pathological diagnosis of oncocytoma was established in all 31 patients. Oncocytoma was present unilaterally in 27 patients and bilaterally in 4. Multiple oncocytomas were found in 5 of 35 affected kidneys. The oncocytomas varied in size from 1 to 13 cm. (mean 3. 7 ) . Focal hemorrhage was occasionally observed within an oncocytoma but none of the tumors dem onstrated capsular penetration or venous invasion. Renal cell carcinoma and renal oncocytoma were found in 10 patients (32 % ) . In 7 patients both tumors were present as separate lesions within the same kidney. In 3 patients renal cell carcinoma and oncocytoma were found in opposite kidneys.
1381
One of the latter patients, initially treated with bilateral partial nephrectomy, had a local tumor recurrence 5 years later in the kidney that had contained renal cell carcinoma. Repeat partial nephrectomy of this kidney revealed the recurrent tumor to be an oncocytoma. Among patients with renal cell carcinoma and oncocytoma, the renal cell carcinoma displayed a granular cell histology in 5, a clear cell histology in 4, and a mixed granular and clear cell histology in 1. One patient was originally diag nosed in 1984 as having renal cell carcinoma developing within a circumscribed oncocytoma. Upon review of the sections in preparation for this article, it was recognized that the foci of marked cellular enlargement, multinucleation and nuclear atypia were as described by Davis et al in 1991 and were characteristic of their cases of atypical oncocytomas with cell ular pleomorphism. 25 After review, this oncocytoma was not considered to have an associated renal cell carcinoma. All other renal cell carcinomas were histologically confirmed to be ma lignancies. Followup was available for 29 patients and ranged from 2 to 122 months (mean 45.6). Of the patients 24 are alive with no evidence of disease, 1 is alive and asymptomatic with residual oncocytoma, 1 with coexistent tumors died of progressive renal cell carcinoma and 3 tumor-free patients died of unrelated diseases. No patient had metastases from oncocytoma. DNA ploidy analysis. Tissue samples from 16 oncocytomas were analyzed for DNA ploidy. In 5 patients the oncocytoma tissue was analyzed by flow cytometry and in 7 it was analyzed by image cytometry. Both methods were used in 4 patients. In all cases the oncocytomas had diploid DNA histograms. HLA class I antigen expression. HLA-A , B, C (class I) antigen expression was determined in 11 renal oncocytomas. Ten on cocytomas showed no HLA-A, B, C antigen expression (see figure) , while 1 showed only trace evidence of expression of these antigens. DISCUS S I O N
Renal oncocytoma is a benign tumor of renal tubular origin. As originally defined by Klein and Valensi, it is a lesion composed of solid sheets of large polygonal eosinophilic cells usually with no cellular anaplasia. 3 Leiber et al described higher grade oncocytomas with the ability to metastasize and cause patient death. 28 However, in their study no patient with a grade 1 oncocytoma experienced metastases or died of the tumor. Davis et al recently suggested that nuclear atypia and cellular pleomorphism are not necessarily evidence for aggressive be havior of an oncocytoma, 25 and we have documented 1 such case as well. Our patient outcome data support the benign nature of this tumor with no instances of metastases and a 100% tumor-specific survival rate at a mean followup of 45.6 months.
A, renal oncocytoma cells show no HLA class 1 antigen expression by immunohistochemical staining. HLA class 1 antigen expression is demonstrated by vascular endothelial cells and macrophages. Reduced from X400. B, renal cell carcinoma demonstrates strong HLA class 1 antigen expression as evidenced by red-orange immunohistochemical reaction product uniformly covering tumor cell membranes. Reduced from X l60.
!: -; :•
. . .. j
1382
RENAL ONCOCYTOMA
On review of the literature, renal oncocytoma is discovered as an incidental finding in approximately 66% of the cases. 29 In our study renal oncocytoma was found incidentally in 21 of 3 1 patients ( 68 % ) during evaluation for a nonurological disor der. Multifocal and bilateral renal oncocytomas were observed in 5 ( 1 6 % ) and 4 ( 1 3 % ) patients, respectively. Renal oncocy toma and renal cell carcinoma have infrequently been found as coexisting lesions within the same 30 or opposite 31 kidney. In a recent review of 1 6 6 patients with oncocytoma, renal cell car cinoma was also present in 7 . 2 % of the patients; in 3 renal cell carcinoma was observed as a small focus within an oncocy toma. 2 5 In our study we found coexisting oncocytoma and renal cell carcinoma in 10 of 31 patients ( 3 2 % ). While this high incidence of coexistence of renal cell carcinoma and oncocy toma may partly reflect the unusual nature of our patient referral population, we believe that this is an important finding with significant clinical implications. It is presently impossible to establish a preoperative diagnosis of renal oncocytoma through radiographic imaging studies. The CT finding of a central stellate scar32 or a spoke-wheel appear ance on angiography33 is not specific and can be seen with renal cell carcinoma. In this study no oncocytoma exhibited a central stellate scar on CT and only 1 tumor had a spoke-wheel pattern on angiography. Some investigators have recommended fine needle aspiration for evaluation of the indeterminant solid renal mass, suggesting that if the cytological findings are con sistent with oncocytoma then conservative surgery3 4 or possibly observation may be warranted. Even if it were possible to establish a prospective diagnosis of oncocytoma, the large num ber of patients in our study with concomitant renal cell carci noma in the same kidney argues against this approach unless a specific indicatio n for nephron sparing surgery is present. 3 5 Many investigators have attempted to differentiate renal oncocytoma and renal cell carcinoma on a cellular and molec ular level. Immunohistochemical staining to determine the tubular cell of origin 1 0- 1 2 and flow cytometry analysis of tumor cell DNA ploidy 1 3 · 1 4 have yielded mixed results. In our study all 16 oncocytomas sampled were diploid, while Rainwater et al found aneuploid DNA in 11 % of grade 1 oncocytomas. 13 Unique chromosomal rearrangement has been found in renal oncocytomas 3 6 and oncocytomas do not display the loss of alleles at loci on the s hort arm of chromosome 3 that is characteristic of renal cell carcinoma. 1 7 Using the anti-renal cell carcinoma monoclonal antibody 5F4, Kochevar and Coffield also showed that higher grade oncocytoma cells stained positive for this renal cell carcinoma specific antigen while grade 1 oncocytoma cells did not. 3 7 We studied the expres sion of HLA-A, B , C or class I antigens on renal oncocytomas as a possible method of differentiating this lesion from renal cell carcinoma. HLA class I antigens are cell membrane -bound proteins that serve as the principal self recognition antigen s in cell-mediated immunity. They are pres ent on almost all nucleated cells and have been shown to be present on normal renal tubular cells. 1 8 Natali et al showed intact HLA class I antigen expression in 9 renal cell carcinomas with no mention of tumor grade, 23 while Kinjo et al demon strated similar antigen expression in 1 1 grade 1 and 2 tumors. 2 4 Data from our laboratory using immunohistochemical staining with monoclonal antibody by the avidin-biotin technique have shown expression of HLA class I antigens by 67 of 68 grades 1 to 3 renal cell carcinomas. 38 Our present results using this same technique in 11 patients demonstrated that 10 oncocytomas did not express HLA class I antigens, while 1 showed only minimal antigen expression. In some tumors cellular loss of HLA class I antigen expres sion has been associated with the presence of malignancy1 9-22 or with progressive tumor dedifferentiation. 2 1 The paradoxical maintenance of HLA class I antigen expression in renal cell carcinoma and loss of such expression in benign renal oncocy tomas are not readily explained. Nevertheless, if these obser-
vations are validated by further experimentation, they may offer an additional method for differentiating oncocytoma from renal cell carcinoma and thereby help to predict tumor behav10r. REFERENCES 1. Hamper!, H.: Benign and malignant oncocytoma.Cancer, 15: 1019, 1962. 2. Hamper!, H.: Oncocytes and oncocytoma. Virch.Arch.Path.Anat., 335: 452, 1962. 3. Klein, M. J. and Valensi, Q. J.: Proximal tubular adenomas of the kidney with so-called oncocytic features. A clinicopathologic study of 13 cases of a rarely reported neoplasm.Cancer, 38: 906, 1976. 4. Harrison, R. H., Baird, J. M. and Kowierschke, S. W.: Renal oncocytoma: ten-year follow-up. Urology, 17: 596, 1981. 5. Merino, M. J. and Livolsi, V. A.: Oncocytomas of the kidney. Cancer, 50: 1852, 1982. 6. Maatman, T. J., Novick,A. C., Tancinco, B. F., Vesoulis, Z., Levin, H. S., Montie, J. E. and Montague, D. K.: Renal oncocytoma: a diagnostic and therapeutic dilemma. J. Urol., 132: 878, 1984. 7. Spring, D. B., Ulirsch, R. C., Starke, W. R. and Brown, S., Jr.: Renal oncocytoma followed for eighteen years without resection. Urology, 26: 389, 1985. 8. Konnak, J. W. and Grossman, H. B.: Renal cell carcinoma as incidental finding. J. Urol., 134: 1094, 1985. 9. Thompson, I. M. andPeek, M.: Improvement in survival of patients with renal cell carcinoma-the role of the serendipitously de tected tumor. J. Urol., 140: 487, 1988. 10. van Krieken, J. H. J. M., Oosterwijk, E., Lycklama a Nijeholt, A. A. B. and Ruiter, D. J.: Renal oncocytoma: a report of two cases with immunohistochemical evaluation. Hum. Path., 19: 460, 1988. 11. Cohen, C., McCue, P. A. and Derose, P. B.: Histogenesis of renal cell carcinoma and renal oncocytoma. An immunohistochemical study. Cancer, 62: 1946, 1988. 12. Eble, J. N. and Hull, M. T.: Glycoconjugate expression in human renal oncocytomas. A lectin histochemical study. Arch. Path. Lab. Med., 112: 805, 1988. 13. Rainwater, L. M., Farrow, G. M. and Lieber, M. M.: Flow cytometry of renal oncocytoma: common occurrence of deoxyribonucleic acid polyploidy and aneuploidy. J. Urol., 135: 1167, 1986. 14. Eble, J. N. and Sledge, G.: Cellular deoxyribonucleic acid content of renal oncocytomas: flow cytometric analysis of paraffin-em bedded tissues from eight tumors. J. Urol., 136: 522, 1986. 15. Hartwick, R. W. J., El-Naggar, A. K., Ro, J. Y., Srigley, J. R., McLemore, D. D., Jones, E. C., Grignon, D. J., Thomas, M. J. and Ayala, A. G.: Renal oncocytoma and granular renal cell carcinoma. A comparative clinicopathologic and DNA flow cy tometric study. Amer. J. Clin. Path., 98: 587, 1992. 16. Kovacs, G., Welter, C., Wilkens, L., Elin, N. and Deriese, W.: Renal oncocytoma. A phenotypic and genotypic entity of renal parenchymal tumors. Amer. J. Path., 134: 967, 1989. 17. Brauch, H., Tory, K., Linehan, W. M., Weaver, D. J., Lovell, M. A. and Zbar, B.: Molecular analysis of the short arm of chro mosome 3 in 5 renal oncocytomas. J. Urol., 143: 622, 1990. 18. Hart, D. N. J., Fuggle, S. V., Williams, K. A., Fabre, J. W., Ting, A. and Morris,P. J.: Localization of HLA-ABC and DR antigens in human kidney. Transplantation, 31: 428, 1981. 19. Nouri, A. M. E., Smith, M. E. F., Crosby, D. and Oliver, R. T. D.: Selective and non-selective loss of immunoregulatory molecules (HLA-A,B,C antigens and LFA-3) in transitional cell carcinoma. Brit. J. Cancer, 62: 603, 1990. 20. Ottesen, S. S., Skouv, J. and Kieler, J.: Reduced HLA-A,B,C expression in tumourigenic v-raf transfected human urothelial cells. Eur. J. Cancer, 26: 305, 1990. 21. Moller, P., Herrmann, B., Moldenhauer, G. and Momburg, F.: Defective expression of MHC class I antigens is frequent in B eel! lymphomas of high-grade malignancy. Int. J. Cancer, 40: 32, 1987. 22. Momburg, F., Ziegler, A., Harpprecht, J., Moller,P., Moldenhauer, G. and Hiimmerling, G. J.: Selective loss of HLA-A or HLA-B antigen expression in colon carcinoma. J. Immunol., 142: 352, 1989. 23. Natali, P. G., Bigotti, A., Nicotra, M. R., Viora, M., Manfredi, D. and Ferrone, S.: Distribution of human class I (HLA-A,B,C)
RE l\,U\.L G :N C O CYTOMA
24. 25. 26.
27. 28. 29. 30. 31.
histocompatibility antigen s in nonnal and rnct11;;,c;m1c tissues cf nonlymphoid origin. Cancer Res . , 44: 4679, Kinjo, T., Takashi, M., Miyake, K. and Nagura, H.: In situ dem onstration of immunological heterogeneity in vascular endothe lial cells of renal cell carcinoma. J. Surg. Oncol., 4 3 : 73, 1990. Davis, C . J., Jr., Mostofi, F. K., Sesterhenn, I. A. and Ho, C . K.: Renal oncocytoma. Clinicopathologica! study of 166 patients. J. Urogen. Path., l: 42, 199 1 . Bauer, T. W . , Tubbs, R . R . , Edinger, M. G . , Suit, P. F . , Gephardt, G. N. and Levin, H. S . : A prospective comparison of DNA quantitation by image and flow cytometry. Amer. J. Clin. Path., 9 3 : 322, 1990. Tubbs, R . R., Sheibani, K., Deodhar, S. D. and Hawk, W . A.: Enzyme immunohistochemistry: review of technical aspects and diagnostic applications. Cleve. Clin. Quart., 48: 245, 1981. Lieber, M . M., Tomera, K. M . and Farrow, G. M.: Renal oncocy toma. J. Urol., 1 2 5 : 481, 1981. Lieber, M . M., Hosaka, Y. and Tsukamoto, T.: Renal oncocytoma. World J. Urol. , 5: 7 1 , 1987. Lehmann, H. D. and Blessing, M. H.: Renal oncocytoma (pathol ogy, preoperative diagnosis, therapy) . Prog. Clin. Biol. Res., 1 00 : 589, 1982. Kavoussi, L. R., Torrence, R . J. and Catalona, W. J.: Renal onco-
32.
33. 34. 35.
36. 37. 38.
]383
with synchronous contralateral renal cell carcinon1a. J. 134: 1 193, 1985. Quinn, M. J., Hartman, D. S., Friedman, A . C., Sherman, J. L., Lautin, E. M., Pyatt, R. S., Ho, C. K., Csere, R. and Fromowitz, F. B . : Renal oncocytoma: new observations. Radiology, 1 5 3 : 49, 1984. Ambos, M. A., Bosniak, M. A., Valensi, Q. J . , Madayag, M. A. and Lefleur, R. S . : Angiographic patterns in renal oncocytomas. Radiology, 1 2 9 : 615, 1978. Cochand-Priollet, B . , Rothschild, E., Chagnon, S . , Nezelof, C . , Debure, A. a n d Galian, A.: Renal oncocytoma diagnosed b y fine needle aspiration cytology. Brit. J. Urol., 6 1 : 534, 1988. Novick, A. C., Streem, S., Montie, J. E., Pontes, J . E., Siegel, S . , Montague, D. K. a n d Goormastic, M.: Conservative surgery for renal cell carcinoma: a single-center experience with 100 pa tients. J. Urol., 1 4 1 : 835, 1989. Walter, T. A., Pennington, R. D., Decker, H. -J. H. and Sandberg, A. A.: Translocation t(9;1 1) (p23;q12): a primary chromosomal change in renal oncocytoma. J. Urol., 1 4 2 : 1 1 7, 1989. Kochevar, J. and Coffield, K. S.: Differential immunostaining of oncocytic renal tumors with an anti-renal cell carcinoma mono clonal antibody. J. Urol., 1 4 1 : 625, 1989. Tubbs, R. R.: Unpublished data.