Anatomy and Pathology of Testis Cancer
Victor E. Reuter
MORPHOLOGIC ANATOMY OF THE TESTIS
The adult testes are suspended by the spermatic cord and located within the scrotum. The testis proper is surrounded by a thick connective tissue layer called the tunica albuginea, which itself is lined by the visceral tunica vaginalis (Fig. 31.1). In the posterior aspect of the gonad is the mediastinum testis that contains blood vessels, lymphatics, nerves, and portions of the rete testis. The testis contains multiple fibrous septae that radiate from the mediastinum testis to the tunica albuginea, and these divide the organ into approximately 250 compartments that contain the seminiferous tubules (1,2). Surrounding the seminiferous tubules is the interstitium that contains Leydig cells, blood vessels, lymphatics, and nerves. Each compartment of the testis contains a maximum of four seminiferous tubules that are very convoluted and that usually empty into the straight portion of the rete (tubuli recti). Each seminiferous tubule is lined by a basement membrane and a thin lamina propria. Within the seminiferous tubule are Sertoli cells as well as germ cells at different stages of differentiation.
Sertoli cells comprise 10% to 15% of cells within the tubule. They are columnar to pyramidal in shape with their long axis perpendicular to the basement membrane (Fig. 31.2). The cytoplasm is granular-eosinophilic and may contain fine vacuoles. The nuclei are round-to-oval with finely granular chromatin and are commonly located within a cell or two of the basement membrane. They contain a prominent nucleolus, the only normal cell within the tubule to do so. Intracytoplasmic Charcot-Bottcher crystalloids are characteristic but are seen preferentially by electron microscopy.
 FIGURE 31.1. Schematic representation is the testis and its adnexae. |
Sertoli cells have phagocytic capacity but also play an important role in regulating spermatogenesis. By immunohistochemistry, they have been shown to express vimentin, cytokeratins 8, 18, and 19, as well as inhibin (3,4). Cytokeratin positivity is routinely observed in immature Sertoli cells, but expression of this intermediate filament is likely in adults in various conditions including testicular atrophy, Sertoli cell tumors (SCT) and in Sertoli cells adjacent to germ cell tumors (GCT), orchitis, and infarct.
Germ cells originate in the yolk sac and migrate to the genital ridge during the first 7 weeks of gestation (5,6). They comprise 85% to 90% of cells within the seminiferous tubule and have the capacity to differentiate (mature). Spermatogonia are undifferentiated cells located adjacent to the basement membrane (Fig. 31.2). They have clear or basophilic cytoplasm, distinct cytoplasmic membranes, small round nuclei with dark chromatin, and no nucleoli. They have the capacity to proliferate and give rise to primary spermatocytes. The latter cells are subclassified into preleptotene, leptotene, zygotene, pachytene, and diplotene spermatocytes based on their nuclear chromatin pattern. These subtle differences are difficult, if not impossible, to discern on routine histologic preparations and irrelevant when evaluating tumor-bearing gonads. In general,
spermatocytes are larger than spermatogonia, have basophilic cytoplasm, indistinct nuclear borders, round nuclei with distinct chromatin patterns, and absent nucleoli (Fig. 31.2). Completion of the first meiotic division gives rise to secondary spermatocytes, which have a short half-life and undergo a second meiotic division to form spermatids. Secondary spermatocytes are smaller than their progenitor cell and have denser chromatin. Spermatids are located toward the lumen of the tubule and have small nuclei with dense chromatin (Fig. 31.2). They transform into spermatozoa through metamorphosis.
spermatocytes are larger than spermatogonia, have basophilic cytoplasm, indistinct nuclear borders, round nuclei with distinct chromatin patterns, and absent nucleoli (Fig. 31.2). Completion of the first meiotic division gives rise to secondary spermatocytes, which have a short half-life and undergo a second meiotic division to form spermatids. Secondary spermatocytes are smaller than their progenitor cell and have denser chromatin. Spermatids are located toward the lumen of the tubule and have small nuclei with dense chromatin (Fig. 31.2). They transform into spermatozoa through metamorphosis.
 FIGURE 31.2. Normal seminiferous tubules in an adult containing spermatogonia (SG), spermatocytes (SY), spermatids (SD), spermatozoa (SZ), and Sertoli cells (SC). A cluster of Leydig cells (LC) is present in the interstitium (H&E stain, 100×). |
Leydig cells are present in the interstitium as single cells or in clusters (Fig. 31.2). Interestingly, they may also be observed in the tunica albuginea, mediastinum testis, epididymis, and even along the spermatic cord, usually intimately associated to nerve bundles (5,7). Leydig cells have abundant eosinophilic cytoplasm and round, regular nuclei with prominent nucleoli. Intracytoplasmic lipofuscin pigment may be seen more commonly in older males. Intracytoplasmic Reinke crystalloids are characteristic of Leydig cells, rarely seen in normal cells, and more commonly observed by electron microscopy where they appear as a hexagonal prism (8,9).
Leydig cells have the capacity to produce testosterone and share an important paracrine function with Sertoli cells (10). They express inhibin but not cytokeratins or vimentin by immunohistochemistry (11).
The rete testis collects the effluent from the seminiferous tubules. It is located within the hilus of the testis and includes the tubulae recti, the mediastinal rete testis, and the extratesticular rete (Fig. 31.1). The tubuli recti are short segments within the septa that connect the seminiferous tubules to the rete testis. The rete testis forms a series of epithelial-lined, interconnecting channels that lead to several dilated vesicular channels comprising the extratesticular rete, which anastomose to give rise to the efferent ducts or tubuli efferentia. The epithelium of the rete is low columnar and exhibits luminal microvilli. Every cell contains a flagellum that is not visible by routine light microscopy. The cells are immunoreactive for cytokeratins as well as vimentin (12).
The efferent tubules have an irregular (undulating) luminal contour. They receive the luminal content from the rete testis and are responsible for resorption of fluid. The epithelial lining is a mixture of ciliated and nonciliated columnar pseudostratified cells that express cytokeratin and variably vimentin by immunohistochemistry. The epithelial cells are surrounded by a thick basement membrane, which in turn is surrounded by a layer of smooth muscle. These tubules lead into the epididymis, a convoluted tubular structure that plays a role in the transport, maturation, and storage of sperm (7) (Fig. 31.1). Transport is aided by a thick smooth muscle layer that surrounds the epididymis. The epididymis is lined by a thick basement membrane and can be divided anatomically into three sections: head, body, and tail, the latter where sperm storage and maturation occurs. The epithelial lining of the epididymis are predominantly tall columnar (principal cells) many of which exhibit stereocilia, but basal cells, clear cells, and luminal cells are also present. The luminal contour of the epididymis is rigid rather than undulating.
The vas deferens arises from the caudal portion of the epididymis, which proximally joins the excretory duct of the seminal vesicles to form the ejaculatory duct. The vas deferens is lined by pseudostratified, columnar epithelium and basal cells, the former containing long stereocilia. The luminal contour of the vas is variably folded, and the epithelium is surrounded by loose connective tissue and a very thick smooth muscle layer.
Several appendages may be encountered on the testis, testicular adnexae, or spermatic cord, the most common being the appendix testis and appendix epididymis (13,14) (Fig. 31.1). The appendix testis is a vestige of the Mullerian duct attached to the tunica vaginalis along the anterosuperior surface of the testis adjacent to the head of the epididymis. It is a small pedunculated structure lined by columnar nonciliated epithelium. The epithelium is undermined by richly vascular connective tissue. The appendix epididymis is a vestige of the mesonephric duct. It is a cystic, pedunculated structure attached to the head of the epididymis. The cyst is lined by low columnar epithelial cells and the external surface is lined by mesothelium. Two other types of appendages are present as incidental findings and represent remnants of mesonephric tubules. They appear as epithelial-lined tubular or cystic structures that are seen along the testicular adnexae or spermatic cord. Depending on their location, they are called vas aberrans or paradidymis (7,14).
TUMORS OF THE TESTIS
Germ Cell Tumors
Testicular GCT comprise approximately 98% of all testicular neoplasms and are the most common malignancy in males between the ages of 15 and 35 years (15) (Table 31.1). They are relatively uncommon; approximately 5,500 to 6,000 new cases will be diagnosed in the United States during this calendar year. Because of their relative rarity, they present a diagnostic challenge to most practicing pathologist. It is remarkable that tumors of such diverse morphology and clinical behavior should be considered as variants of one entity. Nevertheless, there is circumstantial and laboratory evidence to support this practice. First of all, these tumors tend to arise along the axial skeleton, be it the pineal gland, anterior mediastinum, retroperitoneum, or gonads. Second, mixed histologic patterns predominate over tumors with one histologic type. A third compelling piece of evidence relates to the so-called precursor lesion. When these tumors arise in the gonad, irrespective of the morphology, one is likely to identify intratubular germ cell neoplasia (IGCNU), also known in some circles as in situ carcinoma, in the adjacent seminiferous tubules. A fourth important piece of evidence linking these tumors comes to us from genetics, since approximately 80% of tumors, regardless of the primary site and histology, will have at least one isochromosome of the short arm of chromosome 12, which is known
as i(12p). This genetic abnormality is not pathognomonic of germ cell neoplasia, yet it is a very useful diagnostic tool in selected circumstances due to its rare occurrence in other solid tumors (16,17,18).
as i(12p). This genetic abnormality is not pathognomonic of germ cell neoplasia, yet it is a very useful diagnostic tool in selected circumstances due to its rare occurrence in other solid tumors (16,17,18).
TABLE 31.1 WHO HISTOLOGICAL CLASSIFICATION OF TESTIS TUMORSa | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Testicular GCT can be divided into three groups (infantile/prepubertal, adolescent/young adult, and spermatocytic seminoma [SS]), each with its own constellation of clinical histology, molecular and clinical features (19,20). They originate from germ cells at different stages of development. The most common testicular GCT arise in postpubertal men, are characterized genetically by having one or more copies of i(12p), and exhibit other forms of 12p amplification and aneuploidy (21). The consistent gain of genetic material from chromosome 12 seen in these tumors suggests that it has a crucial role in their development. IGCNU is the precursor to these invasive tumors. Their incidence is approximately 6.0 per 100,000 per year with the majority being discovered between 15 and 40 years of age. Several factors have been associated with their pathogenesis, including cryptorchidism, elevated estrogens in utero, and gonadal dysgenesis. Tumors arising in prepubertal gonads are either teratomas or yolk sac tumors (YSTs), tend to be diploid, and are not associated with i(12p) or with IGCNU. The annual incidence is approximately 0.12 per 100,000. SS arises in older patients. These benign tumors may be either diploid or aneuploid and have losses of chromosome 9 rather than i(12p). Intratubular SS is commonly encountered but IGCNU is not. Their annual incidence is approximately 0.2 per 100,000. The pathogenesis of prepubertal GCT and SS is poorly understood.
Intratubular Germ Cell Neoplasia
This term refers to the lesion initially described by Skakkebaek as “carcinoma in situ” (CIS) as well as to other “differentiated” forms of IGCNU (22,23,24,25). Strictly speaking, the lesion originally described by Skakkebaek is now called “Intratubular germ cell neoplasia, unclassified” (IGCNU) by most, at least in the Western Hemisphere.
The story of testicular “CIS”/IGCNU is fascinating and serves as a paradigm for the concept of progression from incipient or preinvasive neoplasia to invasive disease (24,26,27). In 1972, Skakkebaek reported “atypical spermatogonia” in two men undergoing testicular biopsies during a workup for infertility who subsequently developed invasive TGCT. He hypothesized that these cells constituted “CIS.” Two subsequent seminal studies by his group proved that this was indeed the case. In 1978, he reported a series of 555 men who underwent testicular biopsies for infertility (26,27). They identified six patients with evidence of “CIS.” With a median follow-up period of approximately 3 years, three of these patients developed evidence of an invasive germ cell tumor; one of them with bilateral disease. The remaining 449 patients were tumor free during the same follow-up period.
In 1986, the Skakkebaek group reported their experience with contralateral biopsies in 500 patients with unilateral GCT (28). Twenty seven patients (5.4%) were found to have “CIS.” Eight patients received systemic chemotherapy for advanced disease. Of the remaining 19 patients, 7 (37%) developed invasive GCT at this site within the follow-up period. Mathematical modeling suggested that 50% of biopsy-positive cases would develop disease within 5 years. Remarkably, not a single case of contralateral GCT developed in the remaining 463 biopsy-negative patients during the same follow-up period. In a subsequent report, the authors revealed that at least two of the biopsy-positive cases that received systemic therapy subsequently developed contralateral tumors, suggesting that systemic therapy is not always effective against preinvasive disease (29).
It is clear that the original lesion described by Skakkebaek is the precursor to all types of GCT, at least for those that originate in postpubertal gonads, other than SS. In early 1980, a group of distinguished pathologists including Drs. Robert Scully, Juan Rosai, F.K.K. Mostofi, and Robert Kurman met in Minnesota to discuss nomenclature of incipient germ cell neoplasia. They agreed that “CIS” was a poor choice to describe this lesion since it had no features of epithelial differentiation. They suggested the term “IGCNU,” because it was associated with all morphologic types of GCT with the exception of SS. It also underscores the fact that differentiated forms of IGCN may occur, including intratubular embryonal carcinoma.
IGCNU can be seen adjacent to invasive GCT in virtually all cases in which residual testicular parenchyma is present (22,30). As previously mentioned, it is present in up to 4% of cryptorchid patients, in up to 5% of contralateral gonads in patients with unilateral GCT, and in up to 1% of patients biopsied for oligospermic infertility. Its association with TCGT arising in prepubertal patients is still a source of controversy (19,31,32) While some authors suggest that it does not occur,
others state that it does. In either case, we can state with reasonable certainty that, if IGCNU does occur in childhood tumors, it is certainly less apparent.
others state that it does. In either case, we can state with reasonable certainty that, if IGCNU does occur in childhood tumors, it is certainly less apparent.
 FIGURE 31.3. Intratubular germ cell neoplasia. Inset shows atypical cells with irregular nuclei, coarse chromatin, prominent nucleoli, and perinuclear cytoplasmic clearing near the basement membrane (H&E stain, 200×). Background shows IGCN staining for POUF5 (Oct3/4) by immunohistochemistry (100×). |
IGCNU is characterized morphologically by the presence of enlarged, atypical germ cells located immediately above a usually irregularly thickened basement membrane (Fig. 31.3). The atypical cells are either isolated or form a single row along the basement membrane. They are typically larger than spermatogonia, the other cell that usually resides near the basement membrane. IGCNU cells have clear cytoplasm, irregular nuclear contours, coarse chromatin, and enlarged nucleoli, which may be single or multiple. On the other hand, spermatogonia may also have clear cytoplasm but the cells are small, have round and regular nuclear contours, densely packed chromatin, and absent nucleoli (Fig. 31.2). In most cases, tumor-bearing tubules do not have active spermatogenesis and contain mostly Sertoli cells. Sertoli cells may be displaced toward the tubular lumen. Characteristically, they contain a single nucleolus that is small and regular. The nuclei are oval or round with regular borders and the chromatin is fine. The cytoplasm is amphophilic/eosinophilic and not vacuolated.
In essence, the cytologic features of classic IGCNU are those of seminoma. The relationship is supported by the coexpression of a host of histochemical and immunohistochemical markers among both cell types. Further evidence comes from electron microscopy, which has shown that both share common ultrastructural features including the absence of well-developed cytoplasmic intermediate filaments, inconspicuous organelles, glycogen particles, lack of mature desmosomes and cell junctions, and nucleoli with ropy nucleolonema. Tubules whose lumen is filled with these cells may be regarded as “intratubular seminoma.”
TABLE 31.2 IMMUNOHISTOCHEMICAL PROFILE OF TESTICULAR TUMORS | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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IGCNU may extend into the rete testis, usually undermining the epithelium in a “pagetoid” pattern. At times, the epithelium may become hyperplastic and in this setting, it is important not to confuse this finding with the presence of nonseminomatous germ cell tumor.
IGCNU cells contain glycogen and thus are PAS positive, diastase sensitive. Rarely will other intratubular cells, either spermatogonia or Sertoli cells, show similar positivity. Placental-like Alkaline Phosphatase (PLAP) is one of the isoforms of alkaline phosphatase (Table 31.2). PLAP antibodies will stain IGCNU as well as the majority of seminomas and embryonal carcinomas (EC) as well as a smaller percentage of YSTs. Immunoreactivity is seen in virtually all cases of IGCNU and the staining pattern is usually membranous or cytoplasmic. None of the other nonneoplastic intratubular cells are immunoreactive for PLAP, but immunoreactivity may be seen in other types of non-germ cell malignancies (33,34,35,36). C-kit (CD 117) is expressed in a large percentage of IGCN as well as seminomas, but not in other GCT (37). Once again, the staining pattern is cytoplasmic/membranous. Despite the overexpression of this antigen, c-kit is rarely mutated in these tumors. Other antibodies which immunoreact with IGCNU but are rarely used in clinical practice include M2A and 43-F (36,38,39). POU5F1 (Oct3/4) is a very interesting marker which was described earlier in this decade (40). The gene serves as a transcription factor and its product is expressed in pluripotent mouse and human embryonic stem cells and is downregulated during differentiation. Since the gene is also required for self-renewal of embryonic stem cells, knocking out the gene is lethal. Early reports suggest that this antigen is expressed solely in IGCNU, seminoma and embryonal carcinoma, suggesting that these are the types of GCT cells with pluripotency, that is, with capacity to differentiate. In any event, it provides us with yet another marker for IGCNU (Fig. 31.3).
It is important to keep in mind that the presence of neoplastic cells within tubules does not always constitute IGCNU and that one must adhere strictly to the established diagnostic criteria. Besides intratubular seminoma, one can encounter intratubular embryonal carcinoma, intratubular SS, and even metastatic disease such as melanoma and prostatic carcinoma. Intratubular lymphoma and even mesothelioma may also be confused with IGCNU.
Seminoma
Seminomas are the most common GCT arising in the male gonad, whether they arise in a pure state or mixed with other morphologic types (41,42,43,44,45,46,47). “Pure” seminoma account for 27% to 30% of testicular GCT and another 15% to 18% contain syncytiotrophoblasts. Approximately 1% to 2% are
bilateral, which can occur synchronously or asynchronously. Seminomas reach a peak incidence between the fourth and fifth decade of life, which is approximately one decade later than nonseminomatous GCT.
bilateral, which can occur synchronously or asynchronously. Seminomas reach a peak incidence between the fourth and fifth decade of life, which is approximately one decade later than nonseminomatous GCT.
Macroscopic and Microscopic Features.
Seminomas appear as a fleshy, well-circumscribed, yellow-tan mass that, depending on size, may occupy a variable amount of testicular parenchyma or replace it entirely. Areas of necrosis may be observed grossly in up to 20% to 25% of cases. Some seminomas are associated with a granulomatous reaction, and in these cases, the tumor takes on a fibrous and nodular gross appearance. Microscopically, tumor cells are uniform and have round to vesicular nuclei with clear cytoplasm, prominent cytoplasmic membranes, and a centrally-located, round nucleus with a prominent nucleolus (Fig. 31.4). These cells are arranged in sheets or nests separated by thin fibrovascular bands which contain mature lymphoid cells. Mitotic activity is variable. Some cases exhibit extensive fibrosis, particularly those associated with a granulomatous reaction or tumors that have undergone partial regression. Rarely, seminomas may exhibit unusual patterns such as cribriform, pseudoglandular, and tubular growth (48). These do not represent separate entities, but rather histologic variants of classic seminoma. Less than 20% of cases contain syncytiotrophoblasts, but their presence may be associated with focal areas of hemorrhage and necrosis (Fig. 31.4). Seminomas with syncytiotrophoblasts will be accompanied by serum elevation of human chorionic gonadotropin (HCG), but levels will rarely reach above 500 IU/mL (47).
Tumor cells contain glycogen (PAS positive) and express Placental Alkaline Phosphatase (PLAP) and c-kit (CD-117) by immunohistochemistry (33,34,49,50,51,52,53,54,55) but not cytokeratins, CD-30, or inhibin. A minority of seminoma cells may express focal and weak, dot-like or linear immunoreactivity for cytokeratin, however, never diffuse and strong staining throughout the cytoplasm. Like IGCNU, seminoma cells express POU5F1 (Oct3/4) in a nuclear distribution (33,37,56,57) (Table 31.2).
Some seminomas exhibit a significant degree of cytologic atypia (58,59,60,61,62,63,64,65,66), and this fact led to the now abandoned concept of “Anaplastic Seminoma” as a distinct entity with a worse prognosis (67,68). As described, this tumor was characterized by overall morphologic features of a seminoma but containing more pleomorphic cells with nonclear cytoplasm and abundant mitotic figures. Fibrovascular septae and lymphocytes were absent and focal necrosis was commonly seen. This concept did not withstand the test of time since many series later showed that stage for stage, there was no difference in clinical outcome between classic and anaplastic seminomas (43,58,69). In addition, it has become quite evident that mitotic activity in seminomas is quite variable and, in fact, may be quite high even in classical cases (60). Presently, tumors thought to be seminoma but exhibiting atypical histology should trigger consideration of a differential diagnosis of seminoma that includes (a) seminoma with “early carcinomatous differentiation,” (b) solid variants of embryonal carcinoma or YST, (c) lymphoma, (d) sex-cord gonadal stromal tumor, and (e) metastatic disease, including poorly differentiated carcinoma and melanoma. Other causes of atypical histology in seminoma include poor fixation and faulty processing in the pathology laboratory. “Early carcinomatous differentiation” refers to areas of transition from seminoma to embryonal carcinoma. This concept suggests that seminoma cells are not terminally differentiated but rather, under certain poorly understood circumstances, may differentiate into other germ cell tumor-types (47,63,70). The transformed cells can be identified morphologically and confirmed by immunohistochemistry, since they will express markers akin to embryonal carcinoma.
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