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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-0183
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 8 3071-3075
Copyright © 2006 by The Endocrine Society

BRIEF REPORT

Adult Leydig Cell Tumors of the Testis Caused by Germline Fumarate Hydratase Mutations

Luis G. Carvajal-Carmona, N. Afrina Alam, Patrick J. Pollard, Angela M. Jones, Ella Barclay, Noel Wortham, Massimo Pignatelli, Alex Freeman, Sabine Pomplun, Ian Ellis, Richard Poulsom, Mona A. El-Bahrawy, Daniel M. Berney and Ian P. M. Tomlinson

Molecular and Population Genetics Laboratory (L.G.C.-C., N.A.A., P.J.P., A.M.J., E.B., N.W., I.P.M.T.) and Histopathology Unit and In Situ Hybridisation Service (R.P.), London Research Institute, Cancer Research UK, London WC2A 3PX, United Kingdom; Department of Pathology and Microbiology, Division of Histopathology, Bristol Royal Infirmary (M.P.), Bristol BS2 8HW, United Kingdom; Department of Pathology, University College Hospital (A.F.), London WC1E 6JJ, United Kingdom; Department of Pathology, Kings College Hospital (S.P.), London SE5 9RS, United Kingdom; Department of Histopathology, Nottingham City Hospital (I.E.), Nottingham NG5 1PB, United Kingdom; Department of Histopathology, Division of Investigative Science, Imperial College (M.A.E.-B.), London WI2 0NN, United Kingdom; and Department of Pathology, St. Bartholomew’s Hospital (D.M.B.), London EC1A 7BE, United Kingdom

Address all correspondence and requests for reprints to: Dr. Luis G. Carvajal-Carmona, Ph.D., Laboratory of Molecular and Population Genetics, London Research Institute, Cancer Research UK, London WC2A 3PX, United Kingdom. E-mail: luis.carvajal{at}cancer.org.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Leydig cell tumors (LCTs) are the most common non-germ-cell neoplasms of the testis. LCTs are often hormonally active and can result in precocious virilization or in adult feminization. We identified an LCT in an affected individual from a kindred with hereditary leiomyomatosis and renal cell cancer (HLRCC) and a germline fumarate hydratase (FH) mutation (N64T).

Objective: Our objective was to investigate the role of FH mutations in predisposition to LCTs.

Design: We tested for pathogenic effects of the N64T mutation and screened an additional 29 unselected adult LCTs for FH alterations. We also tested these LCTs for mutations in two genes, the LH/choriogonadotropin receptor (LHCGR) and the guanine nucleotide-binding protein {alpha} (GNAS) that had been implicated in LCT tumorigenesis.

Results: No mutations were found in GNAS, and one tumor had a LHCGR somatic substitution. In addition to the HLRCC case with the N64T germline FH mutation, we identified one other LCT with a previously unreported FH mutation (M411I). Both LCTs from these patients showed loss of the wild-type FH allele. Immunohistochemical and in situ hybridization analyses demonstrated activation of the hypoxia/angiogenesis pathway not only in the tumors belonging to the FH mutation carriers but also in several other mutation-negative LCTs.

Conclusions: Our study shows that some LCTs are caused by FH mutations and represents one of the first reports of germline mutations in any type of adult testicular tumor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEYDIG CELL TUMORS (LCTs) of the testis are the most common type of non-germ-cell testicular tumors and represent up to 3% of all neoplasms in this organ (1). Their prevalence can be particularly high in men with gynecomastia or infertility (2, 3, 4, 5). Leydig cells are responsible for testosterone production after the initiation of puberty. LCTs are often hormonally active, leading to virilization in children or to feminization, infertility, and erectile dysfunction in adults (1, 2).

The molecular basis of LCTs is poorly understood. Recent studies have shown that childhood LCTs can be caused by a somatic substitution (D578H) in a mutational hotspot of the LH/choriogonadotropin receptor (LHCGR) gene (6, 7, 8). LCTs with this mutation generally result in a phenotype characterized by isosexual pseudoprecocity. LCTs have also been observed infrequently in individuals of families with the Carney complex, an autosomal dominant syndrome caused by germline mutations in the PRKAR1A gene (9). Mutations in PRKAR1A have, however, been reported in Carney complex patients with large-cell calcifying Sertoli cell tumors but not in any patient with an LCT.

Most LCTs occur in adulthood, and their molecular etiology is less understood than that of childhood LCTs (10). To date, only two studies have investigated molecular changes in adult LCTs. Fragoso et al. (11) found one adult LCT with a somatic substitution in a mutational hotspot (R201C) of the guanine nucleotide-binding protein {alpha} gene (GNAS, also known as gsp); this gene was evaluated in LCTs because of previously reported mutations in hyperplastic gonadal tissue from McCune-Albright syndrome patients. In another study, Giacaglia et al. (12) screened the D578 LHCGR hotspot in two adult LCTs but failed to identify any mutations. Screening of these mutational sites has not been carried out in a large and representative sample of adult LCTs, and thus, their importance in LCT pathogenesis remains unknown. Although it is possible that some adult LCTs result from somatic mutations and from nongenetic predisposing factors such as cryptorchidism and testicular atrophy, reports on familial clustering, co-occurrence with other types of testicular neoplasms, and their presence in disorders of the endocrine system hint at the existence of uncharacterized genetic factors in at least some LCT cases (2, 13, 14, 15).

We have previously shown that mutations in fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC, MIM605839) (16), a syndrome of uterine fibroids, skin leiomyomas, and renal cancers of type II papillary or collecting duct morphology. FH mutations appear to cause tumor growth through activation of the hypoxia pathway (17). We recently identified an LCT in an adult member of an HLRCC family. This man, an FH N64T mutation carrier, previously developed multiple skin leiomyomata and at the age of 55 yr presented with a benign LCT in the left testicle. This observation of a novel tumor type in an FH mutation carrier raised the possibility of a broader phenotypic spectrum in the HLRCC syndrome.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue samples

Owing to the rarity of adult LCTs, archival specimens of tumor and normal material were provided by six hospitals from the United Kingdom. All available cases were ascertained from the histopathology archives at each center. We obtained paraffin-embedded tumor material belonging to the FH mutation carrier from an HLRCC family and from 29 adult LCT cases where there was no reported family history of leiomyomas. Two genitourinary pathologists (M.A.E.-B. and D.M.B.) verified the diagnosis of all tumor samples and highlighted the tumor and normal areas of the sample to be investigated in the molecular study. Of the 30 tumors, none had evidence of calcification or presented bilaterally, five were LCTs with Sertoli cell elements, and 25 had pure LCT histology. Of the 25 pure LCTs, only two had features of malignancy. The median age of tumor presentation was 48 yr (range, 24–79 yr). Our research had full research ethics committee approval.

Mutation screening and loss of heterozygosity

DNA isolation from tumors and matched normal testicular tissue was carried out using standard procedures after manual microdissection. We estimated that all tumor samples contained at least 80% neoplastic cells. The full coding sequence of the FH gene was screened by direct sequencing in forward and reverse orientation and loss of heterozygosity (LOH) analysis was carried out using three microsatellites, including two within FH (experimental details available on request). To genotype the LHCGR D578H mutation, we used methods reported by Liu et al. (7). We genotyped the GNAS R201 hotspot using direct sequencing and the primers reported by Fragoso et al. (11).

Immunohistochemistry (IHC) and in situ hybridization (ISH)

CD34 and the hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) IHC and vascular endothelial growth factor (VEGF) mRNA ISH analyses and scoring were undertaken as reported by Pollard et al. (18). Two independent observers, who were blinded to the tumor’s mutational status (M.A.E.-B. and R.P.), scored all IHC and ISH experiments. We scored CD34 by counting the number of microvessels seen in 10 random high-power fields (HPF). HIF-1{alpha} IHC was scored on the percentage of positively stained nuclei as weak (<25%), moderate (25–50%), strong (50–75%), and very strong (75–100%). VEGF ISH was examined using a dark-field microscope, and signal was scored as relatively weak, moderate, or strong by three independent observers (L.G.C.-C., M.A.E.-B., and R.P.) relative to the staining of the Leydig cells in the normal surrounding testicular tissue. The region of the tumor with the highest expression level was scored.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FH mutations and LOH

Sequence analysis revealed the germline A to C mutation at cDNA position 352 (N64T) in the familial case to be homozygous/hemizygous in the LCT, suggesting loss of the wild-type FH allele (Fig. 1AGo). The mutation was observed in the heterozygous state in the surrounding normal testicular tissue and in DNA isolated from peripheral blood. LOH in the LCT was verified using microsatellites linked to FH (data not shown).


Figure 1
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  		FIG. 1. Mutations and LOH of FH. A and B, N64T mutation (A) and M411I mutation (B) in LCTs (arrows). Wild-type sequence is shown alongside for comparison. Note the absence of the wild-type allele in each case, caused by LOH, as also found by microsatellite analysis (not shown). C, Mapping of the two FH residues (64 and 411, white) mutated in the LCT patients onto the crystal structure of wild-type E. coli FH. Two FH monomers are shown (green and blue) to illustrate the probable effect of the M411I mutation, leading to disrupted interactions between the subunits comprising the FH tetramer. D, Detailed view of the region around residue 411. The location of residue 411 is shown (spacefill view, standard atomic colors). The locations of three other FH mutations found in vivo (16 19 ) are shown in yellow as either spacefill view on the same subunit as M411 (residue 422) or backbone view on the other subunit (residues 322 and 323).

 
We identified an additional FH mutation in one of the sporadic LCTs (a G to A change at cDNA position 1394; Fig. 1BGo). Two intragenic FH microsatellites revealed near absence of one allele, in agreement with loss of the wild-type allele in this tumor. This FH mutation results in a methionine to isoleucine substitution at position 411 (M411I) and has not been previously reported. We did not observe I411 in 140 healthy controls whose DNA we sequenced. Molecular modeling using the Escherichia coli FumC crystal structure showed that M411 is located within a pocket of residues that are important for the dimerization of the FH protein (Fig. 1Go, C and D). A previously reported pathogenic HLRCC mutation (Y422C) (19) also maps to this pocket, and other reported mutated residues are nearby. Interestingly, the M411I mutation carrier had one of the tumors in our collection with histology of LCT with a Sertoli cell component. Owing to ethical restrictions, we were unable to access the clinical history of the M411I carrier or to determine whether this patient (or his relatives) had previously presented with any of the tumors associated with the HLRCC syndrome. We cannot, in any event, exclude the presence of skin leiomyomas, because our experience has shown that they are not always recorded during physical examinations and are often not reported by patients.

LHCGR and GNAS mutations

The screening of the LHCGR mutation hotspot revealed one sporadic LCT, from a 65-yr-old man, that had acquired the D578H somatic substitution. This finding differs greatly from previous reports in children, where a much larger proportion (typically 70–100%) of LCTs have this mutation (7, 8, 12). Our results suggest that the D578H mutation occurs in a small fraction of noninfantile LCTs. The screening of the GNAS R201 hotspot did not reveal any mutations. Our results do not confirm the findings of Fragoso et al. (11), where one of two LCTs examined had mutations at this site. To our knowledge, our study represents the largest systematic screening of these two mutational hotspots in adult LCTs and suggests that they are mutated only in a minority of cases.

Activation of hypoxia/angiogenesis pathway in LCTs

IHC and ISH analyses revealed relatively high expression of HIF-1{alpha} and VEGF in many of the LCTs. The familial (N64T) LCT was the only sample with a strong level of VEGF expression in the study (see Fig. 2Go), and this tumor also showed very strong HIF-1{alpha} expression. Around 25% of the remaining LCTs showed moderate expression of VEGF, and of these, over 85% also showed moderate to strong HIF-1{alpha} expression (data not shown). The M411I FH mutant LCT had the highest expression of HIF-1{alpha} among all the samples examined, showing strong nuclear staining in nearly 100% of the tumor cells (although, because of lack of sufficient tumor material, we were not able to perform VEGF analysis in this case). The LCT with the somatic LHCGR mutation had moderate levels of VEGF but weak HIF-1{alpha} expression. Of the approximately 75% of tumors with weak VEGF expression, over 85% also showed moderate to strong HIF-1{alpha} expression (data not shown). Interestingly, we observed some well-demarcated intratumor heterogeneity in VEGF and HIF-1{alpha} expression (for example, Fig. 2Go); regions of stronger expression generally had a higher cell density, possibly indicative of tumor progression.


Figure 2
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  		FIG. 2. IHC and mRNA ISH in the N64T mutation carrier’s LCT. This figure reveals two regions of tumor separated by a fibrovascular septum. For VEGF, there was clearly far higher mRNA expression (strong) to the left of the septum than to the right (weak). For HIF-1{alpha}, the proportion of positive cells was higher to the left of the septum (very strong) than to the right (strong). The microvessel density shown by CD34 was similar (~150 per HPF) in both regions.

 
Microvessel density, as assessed by CD34 expression, was not significantly different in the two FH mutation carriers’ tumors (median = 112 per HPF; range, 66–150) compared with the other LCTs (median = 155 per HPF; range, 40–570; P = 0.48, Wilcoxon test). The LCT with the somatic LHCGR mutation had relatively low vessel density (62 per HPF). Overall, VEGF and HIF-1{alpha} expression and vessel density did not significantly co-vary (details not shown), suggesting that the hypoxia pathway was not simply being induced in some tumors by physiologically low oxygen levels resulting from suboptimal blood supply.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated that some adult LCTs are caused by inherited FH mutations. Our findings highlight the importance of genetic factors in the etiology of this tumor type, which until now, has generally been assumed to be sporadic in nature. Our results are additionally important and relevant for clinical practice because they demonstrate the existence of LCT cases within multiple neoplasia syndromes such as HLRCC and suggest that hypoxia pathway activation may underlie LCT pathogenesis. Our data are consistent with previous studies showing that normal Leydig cells express VEGF (20) and that expression is often higher in LCTs (5). Furthermore, we have previously shown high expression of HIF-1{alpha} and VEGF in uterine and renal tumors from the HLRCC syndrome (18), consistent with similar findings in LCTs from FH mutation carriers. It appears, however, that activation of the hypoxia pathway also occurs in the development of at least some LCTs without FH mutations, suggesting an overlapping pathogenic mechanism. The presence of skin leiomyomas, which are often subtle, should be sought in all adult LCT patients. FH mutation screening should then be undertaken in such patients. Correspondingly, males in HLRCC families should be warned of the small absolute, but high relative, risk of LCTs and should be advised to undergo regular testicular (self-)examination. Mutations in the FH gene are one of the few known inherited causes of testicular tumors.


   Acknowledgments
 
We are grateful to the In Situ Hybridization Service and the Equipment Park, London Research Institute, Cancer Research UK. We thank members of the HLRCC family for their participation in the study.


   Footnotes
 
Disclosure statement: M.P. is a member of the World Cancer Research Fund advisory board. I.P.M.T. and R.P. receive funding from Cancer Research UK. D.M.B. is supported by The Orchid Appeal. L.G.C.-C., N.A.A., P.J.P., A.M.J., E.B., N.W., A.F., S.P., I.E., and M.A.E.-B. have nothing to declare.

First Published Online June 6, 2006

Abbreviations: FH, Fumarate hydratase; HIF-1{alpha}, hypoxia-inducible factor 1{alpha}; HLRCC, hereditary leiomyomatosis and renal cell cancer; HPF, high-power field; IHC, immunohistochemistry; ISH, in situ hybridization; LCT, Leydig cell tumor; LHCGR, LH/choriogonadotropin receptor; LOH, loss of heterozygosity; VEGF, vascular endothelial growth factor.

Received January 26, 2006.

Accepted May 25, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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