Rete Testis-Associated Nodular Steroid Cell Nests: Description of Putative Pluripotential Testicular Hilus Steroid Cells
Gladell P. Paner, MD,* Glen Kristiansen, MD,t Jesse K. McKenney, MD,¿ and Mahul B. Amin, MD§
Abstract: A putative hilus interstitial cell has been proposed as the cell of origin for testicular tumors of adrenogenital syndrome, but its normal histology is not documented. We present hitherto undescribed nodular steroid cell nests asso- ciated with the rete testis that are distinctive in their morphology and immunohistochemical profile from Leydig cells and do not have the morphology of typical extra-adrenal cortical rests. These nodules measured 1, 1, 1.8, 2, and 2.5 mm in size with a distinct sinusoidal vasculature. Individual cells were rounded to polygonal with evenly distributed moderate-to-abundant eosi- nophilic cytoplasm. The nuclei were homogenous and round, with fine chromatin and occassionally with prominent nucleoli. The differential diagnosis included adrenocortical rests, testicu- lar adnexal Leydig cells, carcinoid tumorlets, paraganglionic rests, and adenomatoid mesothelial proliferation. Immunohis- tochemistry showed positivity for melan A (5/5), inhibin (3/5), and calretinin (2/4), although the immunoreactivity was distinctively different from the concurrent intratesticular Leydig cells and testicular adnexal Leydig cells in all cases. The unique morphology, immunophenotype, and distinctive location of these cells in the testicular mediastinum raises the possibility that these cells represent testicular hilus steroid cells, the putative histogenetic cell implicated for testicular tumors of adrenogenital syndrome. We propose to name these prolifera- tions rete testis-associated nodular steroid cell nests.
Key Words: rete testis, steroid cells, Leydig cells, adrenal rests, ectopic, testicular adnexal Leydig cells, testicular tumor of adrenogenital syndrome, sex cord stromal tumor, immunohis- tochemistry
(Am J Surg Pathol 2011;35:505-511)
From the *Department of Pathology, University of Chicago, Chicago, IL; +Institute of Surgical Pathology, University of Zurich, Zurich, Switzerland; ¿ Department of Pathology & Urology, Stanford University Medical Center, Stanford; and §Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA.
Presented in part at the United States and Canadian Academy of Pathology meeting in San Diego, CA March, 2007.
Correspondence: Mahul B. Amin, MD, Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Suite 8728, Los Angeles, CA 90048 (e-mail: aminm@cshs.org). Copyright @ 2011 by Lippincott Williams & Wilkins
T he prototypic intrinsic steroidogenic cells in the testes are the Leydig cells that are classically situated in the testicular interstitium. The Leydig cells have finely granu- lar eosinophilic cytoplasm attributed to their abundant smooth endoplasmic reticulum producing androgen hormones that are essential for spermatogenesis.19 Inter- stitial Leydig cells may become prominent as a result of hyperplasia and secondary to tubular shrinkage or atrophy such as seen with spontaneous regression of germ cell tumors or age-associated testicular involution.4 It is well recognized that Leydig cells may be seen extraparenchymally as discrete ectopic foci in the sper- matic cord and tunica albuginea, and uncommonly in adventitial tissue between the epididymis and tunica albuginea and epididymis, the rete testis, and vas deferens.14,17,18 These ectopic Leydig cells or testicular adnexal Leydig cells (TALCs) may be seen intimately associated with nerves, blood vessels, or both.17,18 Leydig cell tumor is a sex cord-stromal neoplasm of the testis composed of elements recapitulating the normal develop- ment and evolution of testicular Leydig cells.7,15
Adrenocortical rest cells have been reported in 7.5% of testes in infants.10 These heterotopic adrenocortical nodules histologically recapitulate the cortical zonation of the adrenal gland proper.10 Rarely, these steroidogenic cells may form large testicular tumorous nodules resulting in endocrine overactivity. Testicular tumor of the adrenogenital syndrome is a steroid cell tumor that develops in the setting of elevated adrenocorticotropic hormone (ACTH) levels encountered in adrenogenital syndrome.2,9,22 The histogenesis of testicular tumor of adrenogenital syndrome is debatable and along with adrenocortical rest cells, a putative hilus interstitial cell has been proposed as the cell of origin.22
Herein, we present 5 cases of unique extratesticular nodular steroid cell nests, distinctive from TALCs or adrenocortical rest cells, found incidentally in the region of the rete testis during the evaluation of adult testicular germ cell tumors and chronic epididymitis. The histology, immunohistochemical profile, and differential diagnostic implications are presented.
MATERIALS AND METHODS
Four cases were obtained from the consultation files of one of the authors (M.B.A.) between 2005 and 2010,
| Case | Age (y | Side | Steroid Cell Rest Size (mm) | Main Tumor Size (mm) | Pathologic Diagnosis of Main Testicular Tumor | Main Tumor Stage* | Comment |
|---|---|---|---|---|---|---|---|
| 1 | 35 | Right | 1 | 20 | Pure embryonal carcinoma | pT2 | Rete testis involved by pagetoid ITGCN |
| 2 | 28 | Left | 1 | 30 | Mixed germ cell tumor (embryonal carcinoma 50%; yolk sac tumor 34%; choriocarcinoma 10%; teratoma, mature 1%; seminoma 5%) | pT2 | Rete testis involved by pagetoid ITGCN |
| 3 | 23 | Left | 2 | 10 | Mixed germ cell tumor (embryonal carcinoma 80%; yolk sac tumor 15%; teratoma, predominantly immature 5%; scattered syncytiotrophoblastic giant cells) | pT1 | Rete testis involved by pagetoid ITGCN |
| 4 | 22 | Left | 2.5 | 35 | Mixed germ cell tumor (embryonal carcinoma 80%; yolk sac tumor 10%; teratoma 5%; choriocarcinoma 4%; seminoma 1%) | pT2 | Rete testis involved by pagetoid ITGCN |
| 5 | 58 | Right | 1.8 | N/A | N/A | N/A | Chronic epididymitis and epididymal cyst |
*AJCC 2010 staging.
ITGCN indicates intratubular germ cell neoplasia; N/A, not applicable.
and 1 case was from the surgical pathology file of the University of Zurich, Zurich, Switzerland. Immunophe- notypic characterization included evaluation for melan A, inihibin, calretinin, CD99, synaptophysin, chromogranin, desmin, smooth muscle actin, WT-1, keratin, S100 protein, HMB-45, ACTH receptor and testosterone receptor, and markers associated with sex cord-stromal, neuroendocrine, muscle, paraganglionic, and mesothelial differentiation. After detailed characterization of these
rete testis-associated steroid cell nests, a retrospective review of 74 orchiectomy specimens (60 adults and 14 pediatric patients) from the surgical pathology files of Loyola University Medical Center, Maywood, IL, was carried out. The examination was directed mainly at the testicular mediastinum and rete testis to assess the presence of similar steroid cell nests. Immunohistochem- ical stains for inhibin and calretinin were performed (n = 7) on sections with prominent concurrent clusters of interstitial Leydig cells and TALCs. Immunohistochem- ical staining for inhibin, calretinin, and melan A were performed in 2 testicular tumors of androgenital syn- drome.
CASE REPORTS
Four orchiectomy procedures were performed for testicular tumors and 1 was an epididymal resection for treatment-resistant chronic epididymitis (Table 1). The age of the patients ranged from 22 to 58 years. The 4 testicular tumors were germ cell tumors with embryonal carcinoma as the predominant component in 3 tumors and as a pure constituent in 1 tumor. All 4 tumors were confined to the testis, and intratubular germ cell neoplasia was present in all with pagetoid spread to the rete testis. The nontumoral patient had chronic epididymitis and benign epididymal cyst. All 5 patients had no signs or symptoms attributable to hormonal imbalance.
Histologically, closely associated with the rete testis and situated within the mediastinum of the testis were distinctive circumscribed nodular nests of eosinophilic cells separated from one another by a striking sinusoidal vasculature (Fig. 1). The nodules were unencapsulated and measured 1 to 2.5 mm in size. Although the nests were discrete, they were closely associated with rete epithelium at the periphery. However, in 1 case there was a more intimate association with interstitial growth between rete tubules. The cells were arranged in small nests and short cords or trabeculae of cells, showing prominent retraction artifact at the stromal junction. Individual cells were rounded to polygonal with fine, evenly distributed eosinophilic cytoplasm and indistinct cell borders. The nuclei were round, showed a fine and homogenous chromatin distribution, and showed occa- sionally prominent nucleoli; nuclear grooves were absent. Intracytoplasmic crystalloids of Reinke or endogenous pigment were not identified. Tumor-associated rete testis hyperplasia was apparent in 1 case.
Although the entire spermatogenic maturation sequence was well represented in the 4 orchiectomies, there was decreased spermatogenesis in all 4 uninvolved seminiferous tubules, including widespread tubular sclerosis involving 1 testis. In 3 of 4 testes, interstitial Leydig cells were seen in their typical clusters of 3 to 10 cells and in a few clusters of approximately 20 cells. Larger hyperplastic clusters of >30 Leydig cells were abundant in 1 case. TALCs were seen but were relatively subtle in all 4 testes, intimately associated with nerves (4 of 4) and freely in the stroma (1 of 4). In 3 of 4 testes, both the interstitial Leydig cells and TALCs had a relatively greater amount of cytoplasm than the rete testis- associated steroid cell nests (Fig. 2) and importantly lacked sinusoidal vasculature, trabecular growth, and retraction artifact.
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The immunohistochemical staining reaction for rete testis-associated nodular steroid cell nests and concurrent interstitial Leydig cells and TALCs is summarized in Table 2. Differential staining patterns for melan A, inhibin, and calretinin were seen in the rete testis- associated nodular steroid cell nests versus the interstitial Leydig cells and TALCs of the same testis. Immunor- eactivity to melan A was strong (3+) in the rete testis- associated steroid cells and in contrast was variably absent to moderate (0 to 2+) in the interstitial Leydig cells and was nonreactive to focally (0 to 1+) present in TALCs (Figs. 3A-C). Both the interstitial Leydig cells and TALCs showed strong (3+) expression of both inhibin and calretinin, whereas the rete testis-associated nodular steroid cell nests showed differential absent-to- weak (0 to 1+) inhibin and absent-to-moderate (0 to 2+) calretinin staining in the same testis (Figs. 3D-I). In 1 case, both the interstitial Leydig cells and TALCs were posi- tive for synaptophysin in contrast to rete testis-associated nodular steroid cell nests, which were consistently nega- tive. Other immunohistochemical stains performed includ- ing cytokeratin AE1/AE3, WT1, chromogranin, desmin, smooth muscle actin, S100, CD99, HMB-45, ACTH receptor, and testosterone receptor were all negative in the 3 types of steroid cells; rete testis epithelium was consistently positive for cytokeratin AE1/AE3 and WT-1. The 74 orchiectomy specimens examined included 32 (43%) removed for tumors (24 germ cell tumors, 6 Leydig cell tumors, and 2 other tumors) and 42 (57%) for non-neoplastic conditions. None of the patients had signs or symptoms attributable to adrenogenital syndrome or hormonal imbalance. Histologically, none (0 of 74, 0%) of the testicular mediastinum or rete testes showed similar steroid cell nests described above. We identified TALCs in
| TABLE 2. Summary of Immunoreactivity Patterns of the 3 Types of Testicular Steroidogenic Cells | ||||
|---|---|---|---|---|
| Cases | Immunohistochemical Stain | Rete Testis-associated Nodular Steroid Cells Nest* | Interstitial Leydig Cells* | TALCs* |
| Case 1 | Inhibin | 1+ | 3+ | 3+ |
| Calretinin | 2+ | 3+ | 3+ | |
| Melan A | 3+ | 2+ | 0 | |
| Synaptophysin | N/A | NA | N/A | |
| Case 2 | Inhibin | 0 | 3+ | 3+ |
| Calretinin | 0 | 3+ | 3+ | |
| Melan A | 3+ | 2+ | 1+ | |
| Synaptophysin | 0 | 0 | 0 | |
| Case 3 | Inhibin | 0 | 3+ | 3+ |
| Calretinin | N/A | N/A | N/A | |
| Melan A | N/A | N/A | N/A | |
| Synaptophysin | 0 | 2+ | 2+ | |
| Case 4 | Inhibin | 1+ | 3+ | 3+ |
| Calretinin | 0 | 3+ | 3+ | |
| Melan A | 3+ | 1+ | 0 | |
| Synaptophysin | 0 | 0 | 0 | |
| Case 5 | Inhibin | 1+ | N/A | N/A |
| Calretinin | 2+ | N/A | N/A | |
| Melan A | 3+ | N/A | N/A | |
| Synaptophysin | 0 | N/A | N/A | |
*Immunohistochemical analysis of the 3 types of steroid cells were carried out concurrently on the same section for all 4 cases.
Staining grade: 0 indicates negative; 1+, focal or weak; 2+, moderate; 3+, strong or diffuse; N/A, not available.
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42 of 74 (57%) testis specimens; including 39 of 60 (65%) adult testes and 3 of 14 (21%) pediatric testes. Immuno- histochemical stains with calretinin and inhibin showed similar strong (3+) reactivity in concurrent interstitial Leydig cells and TALCs in the 7 representative cases that were evaluated. Immunohistochemical staining for the 2 testicular tumors of androgenital syndrome were as follows: melan A (2/2, 3+ and 3+), calretinin (2/2, 3+ and 3+), and inhibin (2/2, 3+ and 3+).
DISCUSSION
A broad range of lesions including both neoplastic and pseudotumorous lesions have been described invol- ving, or in close vicinity of, the rete testis. These include epithelial (cysts, rete hyperplasia, adenomatous hyperpla- sia, cystadenoma, rete testis carcinoma), mesothelial (hyperplasia, adenomatoid tumor, mesothelioma), neu-
roendocrine (carcinoid, paraganglioma), and steroid cell proliferations.1,12,23
TALCs, 14,17,18 adrenocortical rest cells,10 steroid cell nests associated with Nelson syndrome and Carney complex, paratesticular Leydig cell tumors, and testicular tumors of adrenogenital syndrome2,9,22 are contempora- rily recognized paratesticular steroid cell proliferations.
Leydig cells are found in the testicular interstitium of adult men between seminiferous tubules, where they usually form small clusters of 3 to 30 cells. Cytologically, Leydig cells are characterized by a round-to-ovoid shape, with a round, often eccentric nucleus with a central nucleolus and abundant finely granular eosinophilic cytoplasm. They may contain large rod-shaped intracy- toplasmic Reinke crystals and lipochrome pigment. Immunohistochemically, interstitial Leydig cells are characterized by the expression of inhibin and calretinin and variable expression of melan A.11,16,30
TALCs are cytologically similar to intratesticular interstitial Leydig cells and commonly occur in the tunica albuginea and adnexal structures in 90% of orchiectomy specimens, often closely associated with blood vessels or small nerves.17,18,20 TALCs in the tunica albuginea may be inconspicuous or rarely form relatively conspicuous nodules of typically scattered groups of 3 to 10 cells without sinusoidal vasculature, whereas those in the rete testis are generally in tight nests of 20 or more cells, frequently with a corded and trabecular architecture. 17 A recent study analyzed 40 orchiectomy specimens and showed positivity for inhibin and calretinin in 92% and 95% of interstitial Leydig cells and in 64% and 86% of TALCs, respectively, thus providing further evidence for the biological similarity of these 2 cell types.14 Similarly, our immunohistochemical staining showed strong (3+) immunoreactivity for inhibin and calretinin in concurrent TALCs and interstitial Leydig cells, which was distinct from the immunoprofile of the rete testis-associated nodular steroid cell nests. The possibility of hilar-situated Leydig cells altered by extrinsic factors such as presence of testicular tumor (eg, compression or ischemia) is also considered. However, it is unlikely that the alteration occurred specifically only in the hilus and not in Leydig cells in the vicinity of the hilum (as shown in Fig. 2) or away from it. Furthermore, 1 case was seen in a patient with chronic epididymitis.
Adrenocortical cells (possibly due to their similar endocrine function) share some similarities with Leydig cells, and may occasionally form notable rests in testicular adnexal structures. Adrenocortical cells also express inhibin and calretinin at higher rates and more commonly express melan A, which is the recommended marker for these cells.6,20,31 At autopsy, adrenocortical rests were found in 15 of 200 (7.5%) testes that were examined, and ranged in size from 0.5 to 7 mm.10 The presence of bona fide adrenocortical rest cells in adult patients without adrenogenital syndrome is rare.22 Histologically, adreno- cortical rests have a smooth outline and a conspicuous connective tissue capsule. The cellular constituents architecturally parallel the adrenal gland proper and show presence of zonation.10
The 5 cases of rete testis-associated nodular steroid cell nests presented here were discrete, conspicuous, unencapsulated nodular clusters with ill-defined borders, architecturally forming nests, cords, or trabeculae, with sinusoidal vasculature and clefting artifact. They did not contain Reinke crystalloids or endogenous pigment and had relatively less prominent cytoplasm compared with their concurrent TALCs and interstitial Leydig cells. Rete testis-associated nodular steroid cell nests showed absent- to-weak inhibin expression, absent-to-moderate calreti- nin, and strong melan A immunostaining in contrast to the concurrent strong (3+) immunostaining for both inhibin and calretinin and absent-to-moderate melan A in TALCs and interstitial Leydig cells in the same testis, underscoring the difference between these cell types. Carcinoid tumorlets are also included in the differential diagnosis because of the trabecular growth pattern,
sinusoidal vasculature, and clefting artifact, however the rete testis-associated nodular steroid cell nests were negative for synaptophysin, chromogranin, and cytoker- atin. The differing immunophenotype in conjunction with the distinct morphology indicates a different histogenesis of rete testis-associated nodular steroid cell nests com- pared with interstitial Leydig cells or TALCs. Whether the presence of germ cell tumors, particularly the presence of pagetoid involvement of rete testis (coincidentally in all 4 cases) has an indirect stimulatory effect for the manifestations of these steroidogenic nests is unclear.
The rete testis-associated nodular steroid cell nests may be considered analogous to the ovarian hilus cells based on their steroid cell morphology and location in the hilar region. However, ovarian hilar cells are morpholo- gically distinct from rete testis-associated nodular steroid cell nests and have a greater resemblance to TALCs on the basis of cytology, their association with hilar veins and nerves, and the presence of crystalloids of Reinke in the cytoplasm.5,24-26,29
Testicular tumors of adrenogenital syndrome are discovered after the clinical diagnosis of adrenogenital syndrome in the majority of cases.2,9,22 Two thirds of these are palpable (up to 10cm), usually discovered in early adult life, the remaining third (usually < 2cm) are discovered in children.22 These tumors are commonly bilateral and multifocal (83% to 93%).2,22 The majority (86%) of the small testicular tumors of adrenogenital syndrome are situated in the testicular hilus and half of the large tumors have some portion interspersed between the rete tubules.22 On the basis of these astute observa- tions, Rutgers et al22 speculated that testicular tumors of adrenogenital syndrome originate from hilar pluripoten- tial cells, which proliferate as a result of the elevated level of ACTH. However, before our study, these cells had not been histologically documented in the human testes. Val et al28 recently reported a novel population of adrenal- like cells in the embryonic and adult mouse testes, distinct from Leydig cells that responded to ACTH. Thus, the investigators propose these cells to be the mouse equivalent of the human ACTH-responsive cells in the testes in congenital adrenal hyperplasia. We assume here that the rete testis-associated nodular steroid cell nests described here, supported by its adrenal-like properties (strong melan A positivity) and rete testis/hilar location, represent proliferations of these postulated but hitherto undescribed hilar pluripotential steroid cells. We per- formed immunostaining on 2 testicular tumors of androgenital syndrome and showed similar strong melan A positivity, although strong calretinin and inhibin staining was also observed. On the basis of the few number of cases studied, a more conclusive association between testicular tumor of adrenogenital syndrome and nodular steroid cell nests cannot be made. None of the 5 patients had symptoms of ACTH overactivity.
From the diagnostic perspective it is important not to confuse these rare rete testis-associated nodular steroid cell nests with other relative more common and more pertinent paratesticular lesions. Lesions within this region
included in the differential diagnosis are carcinoid tumorlets (synaptophysin, chromogranin, keratin posi- tive),21,27 paraganglioma (S100 protein positive sustenta- cular cells),3,12 mesothelial hyperplasia or adenomatoid tumor (calretinin, WT-1, keratin positive),8,13 and rete hyperplasia (WT1 and keratin positive), which can be ruled out based on the observation of characteristic morphologic features and judicious use of immunohis- tochemistry.1,11,23
In summary, we present 5 examples of hitherto undescribed rete testis-associated nodular steroid cell nests that are morphologically and immunohistochemi- cally distinctive from interstitial Leydig cells and TALCs, and do not have the morphology of typical extra-adrenal cortical rests. These pseudotumorous lesions may be mistaken for other pathologic entities occurring at this site. The predilection for association with the rete testis epithelium in the testicular mediastinum raises the possibility that these cells represent testicular hilus steroid cells that are putatively implicated for testicular tumors of adrenogenital syndrome.
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