Somatic von hippel-lindau mutation in clear cell papillary cystadenoma of the epididymis

Somatic von hippel-lindau mutation in clear cell papillary cystadenoma of the epididymis

Somatic von Hippel-Lindau Mutation in Clear Cell Papillary Cystadenoma of the Epididymis MICHAEL Z, GILCREASE, MD, PHD, LAURA SCHMIDT, PHD, BERTON ZBA...

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Somatic von Hippel-Lindau Mutation in Clear Cell Papillary Cystadenoma of the Epididymis MICHAEL Z, GILCREASE, MD, PHD, LAURA SCHMIDT, PHD, BERTON ZBAR, MD, LUAN TRUONG, MD, MICHAEL RUTLEDGE, MD, AND THOMAS M, WHEELER, MD Papillary cystadenoma of the epididymis is an uncommon benign lesion that may occur sporadically or as a manifestation of von Hippel-Lindau (VHL) disease. Neither immunohistochemical studies nor molecular genetic analyses of the VHL gene have been reported previously for this lesion. The authors describe two cases of clear cell papillary cystadenoma of the epididymis, both of which were initially confused with metastatic renal cell carcinoma. Both lesions showed positive immunohistochemical staining for low and intermediate molecular weight keratins (Cam 5.2 and AE1/AE3), EMA, vimentin, arantitrypsin, and arantichymotrypsin. Each was negative for CEA. Because clear cell papillary cystadenoma is similar to renal cell carcinoma histologically, and because both occur as components of the von Hippel-Lindau disease complex, the authors analyzed both cases for the presence of mutations in the VI-IL gene. A somatic VHL gene mutation was detected in one of the two tumors by polymerase

chain reaction followed by singie-strand conformation polymorphism analysis. Direct sequencing revealed a cytosine to thymine transition at nucleotide 694, resulting in the replacement of an arginine with a stop codon after the sixth amino acid of exon 3. As the VHL gene is believed to function as a tumor suppressor gene, VHL gene mutations may play a role in the initiation of tumorigenesis in sporadic cystadenomas of the epididymis. HUM PATHOL 26:1341-1346. Copyright © 1995 by W.B. Saunders Company Key words: papillary cystadenoma, clear cell, epididymis, yon Hippel-Lindau disease, von Hippel-Lindau gene. Abbreviations: VHL, von Hippel-Lindau; ABC, avidin-biotin-peroxidase complex; PCR, polymerase chain reaction; SSCP, strand conformation polymorphism; CT, computed tomography; APMO, adnexal papillary tumor of probable mesonephric origin.

Papillary cystadenoma of the epididymis is an unc o m m o n benign lesion that usually presents as an asymptomatic scrotal massf1-3 Histologically it is composed of multiple cysts and tubules lined by cuboidal to columnar epithelium with papillary fronds protruding into some of the lumina. Some variants exhibit abundant clear cytoplasm and p r o m i n e n t vascularity, and can be confused with metastatic renal cell carcinoma. 4'5 Papillary cystadenoma is t h o u g h t to represent the epididymal c o m p o n e n t of von Hippel-Lindau's (VHL) disease, characterized by retinal and cerebellar hemangioblastomas, cysts of the kidneys, pancreas, and epididymis, and renal cell carcinoma. 6'7 VHL disease exhibits autosomal d o m i n a n t inheritance, and the VHL gene has been recently characterized, s-l"~ It is believed to be a t u m o r suppressor gene, and mutations in both alleles have been identified in renal cell carcinomas and hemangioblastomas. To date, no reports have appeared in which cystadenomas of the epididymis have been examined for

mutations in the VHL gene. In this article, the authors describe two cases of clear cell papillary cystadenoma of the epididymis, and provide immunohistochemical profiles and molecular genetic analyses of the VtIL gene in this u n c o m m o n entity and compare these findings to those e n c o u n t e r e d in renal cell carcinoma. ~4-~5

From the Department of Pathology, Baylor College of Medicine and The Methodist Hospital, Houston, TX; Biological Carcinogenesis and Development Program, Program Resources, Inc/DynCorp National Cancer Institute, Frederick Cancer Research and Development Center, Frederick MD; and Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD. Accepted for publication July 12, 1995. The content of this article does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government. Address correspondence and reprint requests to Thomas Wheeler, MD, The Methodist Hospital, MS205, 6565 Fannin St, Houston, TX 77030-2707. Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2612-000255.00/0

MATERIALS AND METHODS Clinical data were obtained from patient hospital records and from personal communications with the respective clinicians. Tissue sections were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin for routine histological examination. Immunohistochemical studies were performed on formalin-fixed paraffinembedded tissue sections using a modification of the avidin-biotin-peroxidase complex (ABC) method of Hsu et al ~6 and the Vector Elite ABC Kit (Vector Laboratories, Inc, Burlingame, CA). Primary monoclonal antibodies against intermediate cytokeratins (AE1/AE3 cocktail, 1:100, Boehringer Mannheim, Indianapolis, IN), low molecular weight cytokeratin (Cam 5.2, 1:2, Becton Dickinson, San Jose, CA), EMA (1:10, Dako, Carpinteria, CA), vimentin (1:30, Dako), CEA (1:100, Dako Corporation), ch-antitrypsin (1:900, Dako), and cq-antichymotrypsin (1:800, Dako) were used. Paraffin ribbon samples of papillary cystadenomas of the epididymis along with the corresponding normal tissue were obtained from two patients. Paraffin was extracted with noctane, and excess organic solvent was removed with 100% ethanol. Pellets were washed with 70% ethanol, and samples were digested with proteinase K at 55°C for 2 hours, followed by heat inactivation of the enzyme. After centrifugation, the supernatant was passed through a Centricon-100 filter (Amicon, Beverly, MA), and these DNA samples were used directly for mutation analysis.

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TABLE 1.

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PCR Primer Sets for SSCP Analysis of the VHL G e n e

Set

Name

Exon

Sequence of Primers

1

ENIM13E ENR1 MA-8A 10B MA-2A 101 K55 K56 YH1A 6B

1 Sense Axltisense 1 Sense Antisense ] Sense Antisense 2 Sense Antisense 3 Sense Antisense

5'-cctcgcctcgcttacaacag-3' 5'-ttcagggccgtactcttcga-3' 5'-tggtctggatcgcggagggaat-3' 5'~aclgcgattgcagagagtgacctggg-3' 5'-ggcccgtgctgcgctcggtgaact-3' 5'-ccctgctgggtcgggcctaagcgccgggcccgt-3' 5'~gtggctctttaacaacctttgc-3' 5'-cctgtacttaccacaacaaccttatc-3' 5'-ttccttgtactgagaccctagt-3' 5'-taccatcaaaagctgagatgaaacagtgtaagt-3'

2 3 4 5

Samples were analyzed for mutations in the VHL gene by polymerase chain reaction (PCR) followed by single-strand conformation polymorphism (SSCP) analysis.17 Five overlapping sets of PCR primers have been designed to amplify the three exons covering the 852 base pair (bp) coding region of the VHL gene. is The primers used are described in Table 1. PCR reactions were carried out in a 12.5-#L volume containing 2 #L of the extracted DNA; 10 mM Tris-HC1 (pH 8.3); 50 mM KC1; 1.5 mM MgC12; 200 #M each dCTP, d~TP, and dGTP; 30 #M dATP, 12.5 pmoles of each primer, 0.65 units Taq polymerase and 1.25 #Ci sSS-ATP (Amersham, Arlington Heights, IL) in an MJ Research thermal cycler. DMSO (5%) was added to PCR reactions when amplifying exon 1. PCR conditions were as follows: 95°C for 2 minutes followed by denaturation at 95°C for 1 minute, annealing at 62°C for 1 minute, and extension at 72°C for 1 minute for a total of 35 cycles. Reactions were mixed with 6 #L formamide stop buffer (US Biochemical, Cleveland, OH) and heated for 6 minutes at 98°C, then placed on ice. Samples were electrophoresed at 8 W for 16 hours in a 0.5X Hydrolink MDE gel (AT Biochem, Malvern PA) containing 10% glycerol. Gels were dried and exposed to x-ray film for 3 to 5 days. SSCP was confirmed when three i n d e p e n d e n t PCR reactions gave the same SSCP shift. Sequencing reactions were performed in duplicate in both sense and antisense directions, and compared with normal tissue as control. PCR reactions were performed as de-

scribed previously with one biotinylated primer and one nonbiotinylated primer per reaction. The double-stranded DNA product was b o u n d to Dynabeads M-280 Streptavidin (Dynal, Inc, Oslo, Norway) according to the manufacturer's protocol, and the complementary strand was removed by denaturation with 0.15 N NaOH. The streptavidin-biotin-ssDNA complex was used as a template for sequencing by the dideoxytermination method using Sequenase Version 2.0 and the USB protocol. PCR products were subcloned using the TA Cloning Kit (Invitrogen, San Diego, CA) following the manufacturer's protocol. Ampicillin-resistant colonies were checked by PCR to confirm that an insert was present. DNA minipreps were prepared from multiple subclones, and used for SSCP and sequence analysis.

CASE REPORT NO. I A 3- × 4-cm firm n o n t e n d e r right paratesticular mass was noted incidentally during a routine physical examination in a 54-year-old black man with a 10-year history of hypertension. Ultrasound revealed a 1.9- X 1.3- x 1.2-cm inhomogeneous lesion adjacent to the right testicle, and groin exploration was recommended. The patient's serum f l - h u m a n chorionic gonadotropin (fl-HCG) level was noted to be < 5 m I U / m L , and the a-fetoprotein (AFP) was 3.1 ng/mL. Subsequently, a right orchiectomy and epididymectomy were performed. Gross examination showed a 3.3- x 2.6- X 2.5-cm wellcircumscribed encapsulated mass adjacent to but clearly distinct from the testicle. It exhibited a solid tan cut surface with scattered yellow and hemorrhagic foci. Histologically the lesion was comprised of cuboidal and columnar cells with clear cytoplasm lining tubular and cystic spaces, and the intervening stroma was highly vascular (Fig 1A). Only a few of the lumina exhibited papillary fronds (Fig 1B). An oil red O stain showed the cytoplasm to contain abundant lipid. Moderate variation in nuclear size was noted focally, along with occasional small nucleoli. The tumor was completely surrounded by a dense collagenous capsule. The lesion was initially diagnosed as a clear cell tumor suggestive of metastatic renal cell carcinoma, and a workup for a renal neoplasm was recommended.

FIGURE 1. (A) Histological appearance of clear cell papillary cystadenoma from case no. 1, showing tubular and cystic spaces lined by columnar cells with clear cytoplasm and vascular stroma. (Hematoxylin-eosin stain; original magnification x l00.) (B) Some of the lumina exhibit papillary fronds. (Hematoxyiin-eosin stain; original magnification x400.) 1342

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was performed, and the mass was thought to be consistent with an aneurysm. The lesion was surgically excised. Gross examination showed a well-circumscribed, oval, pink-tan nodule measuring 2.5 × 2.2 X 2.0 cm. The external surface was smooth and tan-gray. Sectioning showed the cut surface to be yellow-tan with focal areas of hemorrhage (Fig 2). Histological examination revealed tubules and cysts lined by columnar cells with clear cytoplasm, and the intervening stroma exhibited prominent vascularity (Fig 3A). Occasional papillary fronds were noted within some of the tubular and cystic spaces (Fig 3B). An oil red O stain showed abundant intracytoplasmic lipid. Metastatic renal cell carcinoma was included in the differential diagnosis, and a renal CT scan was performed. No evidence of a renal mass was noted. The lesion was subsequently diagnosed as clear cell papillary cystadenoma of the epididymis.

FIGURE2. Gross a p p e a r a n c e of clear cell papillary cystadenoma from case no. 2, showing a solid yellow-tan cut surface with focal areas of hemorrage.

Renal ultrasound showed a small hypoechoic lobulated lesion in the posterior aspect of the superior pole of the left kidney. A computed tomography (CT) scan showed the lesion to be cystic with slightly irregular margins, along with renal cortical irregularities and focal cortical thinning indicative of prior infarction and scarring. A subsequent renal arteriogram showed splaying of the renal vasculature around the cystic lesion, consistent with a benign cyst. Fine-needle aspiration of the renal cyst produced 2 mL of clear fluid, and cytological examination was negative for malignancy. On reevaluation of the histological and clinical features of the epididymal tumor, a revised diagnosis of clear cell papillary cystadenoma was rendered.

CASE REPORT NO. 2 A 75-year-old man with a history of prostate cancer presented with "a knot in the left testicle." Physical examination showed a 2.8- × 2.5-cm freely movable, pulsatile mass, which appeared to be attached to the left testicle. A Doppler study

IMMUNOHISTOCHEMICAL PROFILES T h e clear cell papillary cystadenomas f r o m b o t h patients showed identical staining profiles. Each s h o w e d positive staining for b o t h low a n d intermediate m o l e c u l a r weight keratins (Cam 5.2 a n d A E 1 / A E 3 ) , EMA, vimentin, oq-antitrypsin, a n d al-antichymotrypsin. Each was negative for CEA (Fig 4A-D).

MOLECULAR GENETIC RESULTS Both t u m o r samples a n d c o r r e s p o n d i n g n o r m a l tissue were e x a m i n e d for V H L g e n e mutations by PCRSSCP using the five overlapping p r i m e r sets that cover the 852 base pair c o d i n g region. T h e t u m o r f r o m case no. 1 showed an a b e r r a n t SSCP b a n d p a t t e r n with p r i m e r set YH1A a n d 6B, which amplifies e x o n 3 o f the V H L gene. This b a n d was n o t p r e s e n t in n o r m a l tissue f r o m the same p a t i e n t (Fig 5). SSCP analysis o f the t u m o r f r o m case no. 2 showed n o alteration in the SSCP p a t t e r n w h e n c o m p a r e d with the n o r m a l tissue for any o f the five p r i m e r sets.

FIGURE 3. Histological a p p e a r a n c e of clear cell papillary cystadenoma from case no. 2 (A) Hematoxylin-eosin stain; original magnification x100; (B) hematoxylin-eosin stain; original magnification ×400. The lesion is morphologically identical to the tumor from case no. 1, 1343

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FIGURE 4. The immunohistochemical profile of the lesion from case no. 1 includes positive staining for (A) cytokeratin. (AE1/AE3 cocktail; original magnification x400,) (B) Epithelial membrane antigen. (Original magnification x400.) (C) Vimentin. (Original magnification x400.) (D) Staining for CEA is negative. (Original magnification ×400.) Identical findings were obtained with the tumor from case no. 2 (Hematoxylin counterstain on all).

Direct sequencing of the t u m o r from case no. 1 was p e r f o r m e d in both sense and antisense directions using primers YH1A and 6B. Sequencing results showed a cytosine to thymine transition at nucleotide 694 (Fig 6). This results in the replacement of an arginine with a stop codon after the sixth amino acid of exon 3. T u m o r samples are usually c o n t a m i n a t e d with

N

T FIGURE 5. SSCP analysis of papillan/ cystadenoma of the epididymis from case no. 1. A typical result is shown. PCR and subsequent SSCP gel electrophoresis were performed using primers YH1A and 6B, which amplify exon 3. Aberrant bands in cystadenoma 14145-1C (case no. 1) are indicated by arrows. The patient's normal tissue is shown on the left.

n o r m a l tissue, resulting in a m i x e d wild t y p e / m u t a n t sequence. By subcloning the PCR product, it is possible to separate wild type and m u t a n t clones to clarify the m u t a t i o n sequence. T h e PCR p r o d u c t o b t a i n e d f r o m amplification o f t u m o r DNA f r o m case no. 1 with e x o n 3 primers was subcloned, and a series o f clones c o n t a i n i n g the 280 base pair insert were subj e c t e d to SSCP analysis and direct sequencing. T h r e e o f the seven subclones were m u t a n t clones, which had the same a b e r r a n t SSCP shift as the original t u m o r DNA f r o m case no. 1. T h e o t h e r f o u r subclones were wild type as indicated by their n o r m a l SSCP pattern. S u b s e q u e n t direct s e q u e n c i n g o f m u t a n t subclones showed a thymine at n u c l e o t i d e 694 c o m p a r e d with a cytosine in the same position in subclones with normal SSCP pattern.

DISCUSSION Neither molecular genetic analyses n o r immunohistochemical profiles have previously been r e p o r t e d for papillary cystadenomas of the epididymis. However,

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Numerous other types of sporadic tumors have been examined without evidence of VHL gene involvem e n t . 13,19

T

C to T - - ~

ACGT

T A G C G A G A A

Interestingly, the same VHL gene mutation identified in the sporadic clear cell papillary cystadenoma in this report has been found at nucleotide 694 in four VHL patients. 2°'z3 This mutation is the third most frequent VHL gene mutation identified to date after those occurring at nucleotides 712 and 713. The identification of a VHL gene mutation in a papillary cystadenoma provides a possible explanation for the increased frequency of this lesion in VHL disease. Demonstration of loss of heterozygosity in papillary cystadenomas from VHL patients would provide further support for VHL gene involvement in the initiation of tumorigenesis in papillary cystadenomas.

?

REFERENCES

N

ACGT

FIGURE 6. Sequencing analysis of exon 3 PCR products in papillary cystadenoma 14145-1C (case no. 1). Cystadenoma (left) and normal tissue (right) are shown. Right ordinate, normal sequence. Tumor has a C to T transition at nucleotide 694.

the immunohistochemical profile for a benign adnexal papillary t u m o r of probable mesonephric origin (APMO) has recently been reported. 1"9 This lesion is thought to be the female counterpart of papillary cysta d e n o m a o f the epididymis. 2° APMO has been shown to be cytokeratin and EMA positive and vimentin negative, in contrast to the papillary cystadenomas in this study, which were positive for all three markers. Because clear cell papillary cystadenoma closely resembles renal cell carcinoma histologically, and because both are c o m p o n e n t tumors observed in VHL disease, the authors sought to determine whether sporadic clear cell papillary cystadenoma exhibits a VHL gene defect. T h e VHL gene is present on the short arm of c h r o m o s o m e 3, and it has been m a p p e d to 3p25-p26 by genetic linkage analysis. Patients with VHL disease possess one wild-type allele and one mutated allele. Somatic mutations that lead to loss of the wild-type allele in VHL patients have been associated with the developm e n t o f renal cell carcinoma, hemangioblastoma, pheochromocytoma, and pancreatic tumors. 8 VHL gene mutations have also been detected in sporadic renal cell carcinomas 11'13'21 and hemangioblastomas. 22 This gene is thought to act as a tumor suppressor gene. It is believed that inactivation o f both alleles o f the VHL gene is necessary for initiation of tumorigenesis in these tumors. Papillary cystadenomas of the epididymis occur with increased frequency in VHL patients, and the presence of bilateral epididymal cystadenomas in particular is highly suggestive of VHL disease. 3 Clear cell papillary cystadenoma of the epididymis is now the third type of sporadic t u m o r identified with a VHL gene mutation.

1. Price EB, Jr: Papillary cystadenoma of the epididymis: A cling copathologic analysis of 20 cases. Arch Pathol 91:456470, 1971 2. Meyer JS, Roth LM, Silverman JL: Papillary cystadenomas of the epididyInis and spermatic cord. Cancer 17:1241-1247, 1964 3. Tsuda H, Fukushima S, Takahashi M, et al: Familial bilateral papillary cystadenoma of the epididymis: Report of three cases in siblings. Cancer 37:1831-1839, 1976 4. Thoenes W, Storkel S, Rumpelt HJ: Histopathology and classification of renal cell tumors (adenomas, oncocytomas and carcinomas) : The basic cytological and histopathological elements and their use for diagnostics. Pathol Res Pract 181:125-143, 1986 5. Holthofer H, Miettinen A, Paasi~lo R, et al: Cellular origin and differentiation of renal carcinomas: A fluorescence microscopic study with kidney-specific antibodies, intermediate filament antibodies, and lectins. Lab lnvest 49:317-326, 1983 6. Melmon KL, Rosen SW: Lindau's disease: Review of the literature and study of a large kindred. A m J Med 36:595-617, 1964 7. Hortun WA, Wong V, Eldridge R: Von Hippel-Lindau disease: Clinical and pathological manifestations in nine families with 50 affected members. Arch Intern Med 136:769-777, 1976 8. Crossey PA, Foster K, Richards FM, et al: Molecular genetic investigations of the mechanism of tumorigenesis in yon Hippel-Lindan disease: Analysis of allele loss in VHL tumors. Hum Genet 93:5358, 1994 9. Latif F, Tory K, GnarraJ, et al: Identification of the yon Hippel-Lindau disease tumor suppressor gene. Science 260:1317-1320, 1993 10. Seizinger BR: Toward the isolation of the primary genetic defect in yon Hippel-Lindau disease. Ann NYAcad Sci 615:332-337, 1991 11. Foster K, Crossey PA, Cairns P, et al: Molecular genetic investigation of sporadic renal cell carcinoma: Analysis of allele loss on chromosomes 3p, 5q, l l p , 17, and 22. BrJ Cancer 69:230-234, 1994 12. Seizinger BR, Smith DI, Filling-Katz MR, et al: Genetic flanking markers refine diagnostic criteria and provide insights into the genetics of von Hippel-Lindau disease. Proc Nail Acad Sci USA 88:2864-2868, 1991 13. Gnarra JR, Tory K, Weng Y, et al: Mutations of the VHL tumor suppressor gene in renal carcinoma. Nature Genet 7:85-90, 1994 14. Mackay B, Ordonez NG, Khoursand J: The ultrastructure and immunocytochemistry of renal cell carcinoma. Ultrastruct Pathol 11:483-502, 1987 15. Oosterwijk E, Ruiter DJ, WakkaJC, et al: Immunohistochemical analysis of monoclonal antibodies to renal antigens: Applications in the diagnosis of renal cell carcinoma. Am J Pathol 123:301-309, 1986 16. Hsu S-M, Raine L, Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC method and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:57%580, 1981

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17. Orita M, Suzuki T, Sekiya T, et al: Genomics 5:874-879, 1989 18. Chen F, Kishida T, Yao M, et al: Germline mutations in the von Hippel-Lindau disease tumor suppressor gene: Correlation with phenotype. Hum Mut 5:66-75, 1995 19. Gaffey MJ, Mills SE, Boyd JC: Aggressive papillary tumor of middle ear/temporal bone and adnexal papillary cystadenoma. Am J Surg Pathol 18:1254-1260, 1994 20. Crossey PA, Richards FM, Foster K, et al: Identification of intragenic mutations in the yon Hippel-Lindau disease tumor suppressor gene and correlation with disease phenotype. Hum Mol Genet 3:1303-1308, 1994

21. Shuin T, Kondo K, Torigoe S, et al: Frequent somatic mutation and loss of heterozygosity of the von Hippel-Lindau tumor suppressor gene in primary human renal cell carcinomas. Cancer Res 54:2852-2854, 1994 22. Kanno H, Kondo K, Ito S, et al: Somatic mutations of the von Hippel-Lindau tumor suppressor gene in sporadic central nervous system hemangioblastomas. Cancer Res 54:4845-4847, 1994 23. Loeb DB, Pericak-Vance MA, StajichJM, et al: A novel mutation in the von Hippel-Lindau gene. Hum Mol Genet 3:1423-1424, 1994

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