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Testicular Cancer Pathogenesis, Diagnosis and Endocrine Aspects

, MD, PhD, DMSc, , MD, DMSc, and , MD, PhD.

Author Information

Last Update: January 7, 2018.

Abstract

Testicular cancer comprises different neoplasms, depending on the cell of origin and the typical age at presentation, but germ cell-derived tumors constitute the vast majority of cases. Germ cell tumors can be diagnosed in every age group, but more than 90% of cases occur in young men. These tumors, comprising seminoma and nonseminoma, are derived from germ cell neoplasia in situ (GCNIS). Pathogenesis of testicular tumors associated with GCNIS partly overlaps with that of other developmental disorders of the male reproductive system, within the proposed testicular dysgenesis syndrome (TDS). Testicular somatic cell tumors, known as sex cord-stromal neoplasms, are relatively rare, but can have endocrine manifestations, such as precocious puberty or gynecomastia. In addition to its malignant features, cancer of the testis represents a developmental, endocrine and reproductive problem. These issues are in focus in this chapter, and emphasis is given to aspects that are of interest to endocrinologists, including pediatric endocrinologists and andrologists. Management of invasive testicular tumors is largely handled by urologists and oncologists, thus only general information on surgical treatment and chemotherapy is presented. Impact of treatment on endocrine system, co-morbidity, fertility issues and quality of life issues are also briefly reviewed.

TAKE HOME POINTS

1-Testicular germ cell tumors (TGCT) may occur in any age but >90% occur in young adult men, with the peak prevalence between 25 and 40 years of age. These tumors are derived from germ cell neoplasia in situ (GCNIS) cells, which are arrested and transformed fetal gonocytes that failed to mature to spermatogonia.

2-The presence of GCNIS should be suspected in men with subfertility, impaired spermatogenesis or history of cryptorchidism. Many cases may be linked together under the concept of the testicular dysgenesis syndrome (TDS).

3-Increasing worldwide incidence, but with marked ethnical and geographic differences, is consistent with a predominant importance of environmental/life style factors but also genetic susceptibility.

4-Testicular tumors derived from somatic cells are rare and occur predominantly in childhood. These tumors have mainly benign clinical course, so testis-sparing surgery should always be considered.

5-Bilateral testicular nodules are almost always adrenal rests caused by congenital adrenal hyperplasia and must be managed conservatively to avoid erroneous orchidectomy.

6-TGCTs are treated predominantly by surgery, followed by chemotherapy regimens and/or radiotherapy depending upon histology, disease spread and other prognostic factors.

7-Andrological management, including sperm cryopreservation and monitoring of reproductive hormones, is essential in patients with testicular cancer, who are at a high risk of infertility and hypogonadism.

INTRODUCTION

Testicular cancer comprises a number of different neoplasms, depending on the cell of origin and the typical age at presentation (1, 2). Although several cell types in the testis can undergo neoplastic transformation, germ cell-derived tumors constitute the vast majority of cases of testicular neoplasms. The relative distribution depends on age: whereas in young adult men nearly all tumors are germ cell tumors, in patients over 70 years old, a large proportion of lymphomas and secondary carcinomas can be expected (2). In other words, as explained in detail further in the text, germ cell cancer can be diagnosed in every age group, but more than 90% of cases occur in young men, and this subgroup is the main focus of this chapter. Pathogenesis of testicular germ cell tumors of young adults partly overlaps with that of other developmental disorders of the male reproductive system, within the proposed testicular dysgenesis syndrome (TDS) (3, 4, 5). Somatic cell tumors in the testis, known as sex cord-stromal neoplasms are relatively rare, but are also discussed in this chapter, because being derived from endocrine active cells, they have endocrine manifestations.

In addition to its malignant features, cancer of the testis represents a developmental, endocrine and reproductive problem. These issues are in focus in this chapter, and emphasis is given to aspects that are of interest to endocrinologists, including pediatric endocrinologists and andrologists. Management of overt testicular tumors is largely handled by urologists and oncologists, thus only general information on surgical treatment and chemotherapy options is presented here. However, the contribution of andrologists and endocrinologists to the management of patients with testicular cancer is very important, especially concerning early diagnosis, fertility issues, hypogonadism and the impact of treatment on the quality of life of the patient.

GERM CELL TUMORS (GCT)

Germ cell tumors (GCT) are characterized by extreme phenotypic heterogeneity. They display features of pluripotency and about half of them can differentiate into virtually any somatic tissue type and form teratomas that recapitulate early embryonic differentiation (6, 7). Although in comparison to other solid tissue cancers, GCTs are relatively rare, they constitute the most common form of solid tissue cancer of young age. The typical localisation of GCT is the testis in males and ovary in females, but GCTs may also be found outside the gonads, thus these tumors are named extragonadal GCTs (8, 9). Extragonadal GCTs occur most often in children of both genders, preferentially along the body midline (intracranial, pineal, mediastinal) but can occur also in adults. An increased frequency of extragonadal GCTs, especially in mediastinum, has been associated with the Klinefelter syndrome (10). It is important to remember that a large proportion of cases of extragonadal GCT in retroperitoneal locations are associated with pre-malignant changes in testicles, and can be assumed early metastases of testicular neoplasms, although a multi-site development of GCT cannot be completely excluded (8, 11, 12) This chapter focuses on the testicular GCT.

Classification, Histopathology and Phenotype of Testicular Germ Cell Tumors (TGCT)

Testicular germ cell tumors (TGCT) are by far the most frequent neoplasms of the testis and comprise approximately 90-95% of cases. They may affect infants (rarely), young men (commonly) and elderly men (rarely). The rare TGCTs of infants and elderly are mentioned only briefly at the end of this section.

The most commonly accepted and currently used is the WHO classification, last updated in 2016. After years of discussions and parallel attempts, the latest WHO proposal is based on patho-biological evidence (2). The most important change is the new division of TGCT into two major groups according to the origin from germ cell neoplasia in situ (GCNIS). For use by non-pathologists, a simplified division shown in Table 1 is sufficient.

Table 1Simplified classification of testicular germ cell tumors (TGCT), based on WHO classification from 2016 (2).

· GCT derived from germ cell neoplasia in situ (GCNIS)

o GCNIS (9064/2)*

o Seminoma, pure (9061/3)

    • Non-seminomatous GCT, pure

- Embryonal carcinoma (9070/3)

- Yolk sac tumor, postpubertal type (9071/3)

- Trophoblastic tumors, incl. choriocarcinoma (9100/3)

- Teratoma, postpubertal type (9080/3)

    • Non-seminomatous mixed germ cell tumors (9085/3)

o Regressed GCTs (9080/1)

  • GCT unrelated to GCNIS

o Spermatocytic tumor (9063/3)

o Prepubertal (pediatric) tumors

- Teratoma, prepubertal type (9084/0)

- Yolk sac tumor, prepubertal type (9071/3)

- Mixed prepubertal type tumors (9085/3)

*Footnote to Table 1: The codes in parentheses are from the International Classification of Diseases for Oncology (ICD-O).

Precursor Lesions

The TGCT of young adults (seminoma and nonseminoma) originate from a common precursor, germ cell neoplasia in situ (GCNIS), initially termed carcinoma in situ (CIS) testis (13, 14, 1).

GCNIS is considered to originate from developmentally arrested immature germ cells (gonocytes) that persisted outside of fetal/perinatal life (4, 14, 15). Accordingly, morphology of GCNIS cells resembles closely that of fetal gonocytes, but with more irregular chromatin in the nuclei. GCNIS cells are located inside seminiferous tubules, most frequently in a single row along the basement membrane (Figure 1).

Gonadoblastoma is a preinvasive lesion which occurs almost exclusively in individuals with disorders of sexual development (DSD). This lesion is most often found in patients with mixed gonadal dysgenesis (45X/46XY) (2, 16, 17, 18). Gonadoblastoma cells and GCNIS cells have a very similar gonocyte/oogonium-like phenotype, but the surrounding somatic cells are different; GCNIS cells are present inside seminiferous tubules (which are usually well developed but may be hypoplastic and contain immature Sertoli cells), while gonadoblastoma consists of groups of cells, which are nested in small stromal cells similar to granulosa cells (17, 18, 19, 21, 22). For this reason, gonadoblastoma has been classified by WHO as a neoplasm comprising both germ cells and somatic cells (2). However, GCNIS and gonadoblastoma can be present in the same gonad and there are lesions in between both entities (16, 17). The clinical course of pure gonadoblastoma may be benign, but it has a potential to transform into a malignant germ cell tumor, especially if accompanied by GCNIS and greater virilization (17, 18, 22).

Image testi-canc-pathogen_fig-1.jpg

Figure 1. Histology of germ cell neoplasia in situ (GCNIS)

The upper panel (hematoxyllin-eosin, HE staining shows a low magnification view of GCNIS in a typical pattern with only GCNIS cells and Sertoli cells present inside tubules. The tubules with neoplasia have a smaller diameter than normal seminiferous tubules. On the right side of this image a few tubules with decreased spermatogenesis are visible . The lower left image shows a fragment of a tubule with GCNIS side-by-side with a tubule with preserved spermatogenesis; note the large GCNIS nuclei. The lower right image displays GCNIS cells visualised by immunohistochemical staining for placental-like alkaline phosphatase (PLAP).

The immunohistochemical and gene expression profiles of GCNIS and gonadoblastoma cells are virtually identical and resemble very closely those of primordial germ cells and fetal gonocytes (19, 23, 24). These data were subsequently confirmed by comparative studies at the transcriptional level using microarrays (15, 25, 26). Among many genes highly expressed in GCNIS cells (as well as gonadoblastoma, normal fetal germ cells and overt TGCTs) the following should be mentioned because of their interesting biological function (e.g. germ cell survival or maintenance of embryonic stem cell pluripotency) and usefulness in histopathological diagnosis (24): KIT (23, 27), P53 (28), OCT4 (29), AP2-gamma/ TFAP2C (25, 30), NANOG (25, 31, 32), and LIN28 (33, 34).

In addition to protein-coding genes, GCNIS cells display a specific profile of embryonic-type micro-RNAs (miRNA); miR-371-3 cluster, miR-302 and miR-367 (35). This miRNA profile is also expressed in overt TGCT, except teratoma (see section on serum markers below).

Testicular Germ Cell Tumors (TGCT) Derived From GCNIS: Seminoma and Nonseminoma

As mentioned in the Introduction, TGCT of young adult men display very variable histology (some examples are shown in Figure 2) and are divided into seminoma and nonseminoma (2).

Image testi-canc-pathogen_fig-2.jpg

Figure 2. Histology of main types of testicular germ cell tumors.

The large images show a general histology pattern of a seminoma (upper panel) and two most often seen types of non-seminoma: undifferentiated embryonal carcinoma (middle panel) and teratoma, a tumor displaying differentiation into various somatic tissues (bottom panel). Small square pictures on the right show cellular characteristics in a greater magnification. All sections are stained with hematoxyllin-eosin (HE).

Seminomas are most often diagnosed in the 25- to 40-year-old age group, whereas nonseminomatous tumors occur in even younger men (adolescence to 30 years). Both types originate from GCNIS (14, 2). Seminoma resembles a mass of immature germ cells; the tumor cells are morphologically very close to GCNIS cells and proliferate as a homogeneous tumor, which retains features of germinal lineage. The gene expression profile of seminoma is similar to that of GCNIS and fetal gonocytes, and virtually identical to the female equivalent, called dysgerminoma (24, 26, 36, 37).

Nonseminomatous tumors display a variety of histological forms and contain undifferentiated embryonal carcinoma and somatic components partly differentiated along embryonic lineage of any tissue type (1, 2). Nonseminomas also contain extra-embryonic tissue components (yolk sac tumor and choriocarcinoma). The combined or mixed tumors contain elements of seminoma and nonseminoma but are classified and clinically treated as nonseminoma, which usually has more serious clinical course than seminoma (1, 2).

Testicular Germ Cell Tumors Not Associated With GCNIS

Prepubertal (pediatric) TGCTs: These tumors occur in early childhood (between birth and approximately 5 years of age) and comprise two histological types: yolk sac tumor of the prepubertal type and mature teratoma (including dermoid cyst). The histology of the prepubertal tumors does not differ from the adult equivalents (components of nonseminoma) (2). Likewise, the components display similar characteristic transcriptome and micro-RNA profiles (38, 39). The etiology and pathogenesis of infantile TGCT remain unknown. These tumors are assumed to originate from primordial germ cells (PGC) but there is no known precursor lesion of GCNIS/gonadoblastoma type (2, 40, 41).

Spermatocytic tumor: This rare tumor occurs only in testes of older men (median age at diagnosis around 50 years) and has no extragonadal or ovarian counterpart. This tumor is not derived from GCNIS and has a gene profile similar to spermatogonia or early primary spermatocytes (24, 43, 44, 45). Spermatocytic tumors appear to grow from clonally expanding spermatogonia. The following de novo genetic aberrations causing increased spermatogonial proliferation have been identified; amplifications in chromosome 9p encompassing DMRT1 locus (43), rare gain-of-function mutations in genes encoding FGFR3, HRAS, NRAS, and simultaneous whole chromosome gains of chr9 and chr20 combined with a loss of chr7 (46, 47). Some of these mutations that occur spontaneously in germ cells of aging men, if transmitted to next generation, can cause severe skeletal inborn abnormalities, such as achondroplasia, thanatophoric dysplasia or Costello syndrome (48).

Etiology and Pathogenesis of Testicular Germ Cell Cancer of Young Adults (Derived From GCNIS)

Incidence Trends and Risk Factors

Epidemiology of testicular germ cell tumors (TGCT) has attracted growing attention of researchers, because of the steadily rising incidence (49, 50, 51). The incidence is remarkably variable geographically and dependent on the ethnic background (Figure 3). It is currently the most common malignancy of young men of Caucasian origin, while it is not common among Asians and Africans (50, 51, 52). Interestingly, indigenous Maoris of New Zealand have a higher incidence of testicular cancer than the white population (53).

Image testi-canc-pathogen_fig-3.jpg

Figure 3. Global incidence rates of testicular cancer.

Age-standardized incidence rates for testicular cancer for men of all ages in selected countries of all regions of the world, extracted from national registries. National reporting systems varied by country, and data quality may have fluctuated between regions. Reprinted from Znaor et al., Eur Urol 2014 (51).

However, the incidence rates are not stable and change over time. Within populations rapid changes have been observed in recent decades. For example, there is a nearly three-fold higher incidence in Norway and Denmark in comparison to a nearby Finland. But while the incidence rate in Denmark since 1970-s has been attenuating, an alarming rise has been recently noted in Finland and in Slavic countries, especially Slovakia, Slovenia and Croatia (52, 54, 55). Similarly, a rapid increase has been noted among the Hispanic population in the United States (56). Another interesting feature of epidemiology of testicular cancer is the so-called birth cohort effect; the rise in the incidence correlated with the calendar year of birth rather than with the age at diagnosis (57). For unexplained reasons, cohorts born at wartime had lower incidence than men born just before or after the war (57).

As mentioned before, testicular cancer has an unusual age-specific incidence rate with a small peak in the postnatal period and a major peak in young adult age, starting at puberty. These periods coincide with an activation of gonadal hormones, indicating there may be a possible connection between the hormonal factors and invasive transformation of germ cells. In certain risk groups, the incidence of testicular cancer is much increased. Individuals with developmental abnormalities of the gonads and sex differentiation (DSD) are at high risk of developing germ cell neoplasia. Among these, individuals with the so-called mixed gonadal dysgenesis and the 45,X/46,XY karyotype, and with a partial androgen insensitivity syndrome, are at particular risk of harboring GCNIS or gonadoblastoma and developing TGCT in adolescence (16, 18, 22, 58, 59). An association with testicular cancer has been noted in a number of developmental abnormalities, such as cryptorchidism (60, 61), Down syndrome (62) but also low birth weight and unspecific perinatal factors, e.g. premature birth, birth order, high levels of maternal estrogens or bleeding during pregnancy, high maternal age and neonatal jaundice (61, 63). A late age at puberty and tall stature are also associated with lower and higher risk of testicular cancer, respectively (64, 65). Male infertility is one of the commonest risk factors for testicular cancer and men with testis cancer had poorer semen quality and significantly fewer children than controls prior to development of their tumor (66, 67, 68), see also the section on testicular dysgenesis syndrome (TDS) below.

Patients with a unilateral TGCT are also at an increased risk to develop a new primary testicular tumor of the contralateral testis. Depending on the studied population and the interval from the primary diagnosis, approximately 2 - 8% patients will be diagnosed with a second TGCT during their lifetime (69, 70, 71). The majority of bilateral TGCT occur metachronously, some as late as 20-40 years after the primary TGCT (72). The histology of the primary tumor influences the risk only marginally, with the predominance of seminomas among the synchronous TGCTs but primary non-seminomas observed most often in younger patients with metachronous tumors developing after shorter intervals (69, 70). The presence of testicular atrophy increases the risk of bilateral neoplasia considerably (73, 74).

Testicular Dysgenesis Syndrome (TDS)

The association of testicular cancer with poor testicular function, cryptorchidism, hypospadias and abnormal testicular differentiation led to a hypothesis that poor gonadal development and testicular neoplasia are etiologically linked. A concept of testicular dysgenesis syndrome (TDS) was proposed, in which testicular cancer is one of the symptoms (3, 5). This hypothesis is supported by histological studies showing that dysgenetic features, such as undifferentiated tubules with immature Sertoli cells, clusters of poorly differentiated tubules, or hyaline bodies, often seen in association with testicular cancer, are not uncommon among men referred to andrology clinics because of fertility problems (3, 5, 17, 71). It is important to underline here that not all cases of genital malformations and infertility are a part of TDS, only cases linked to poor development of the testis in fetal life can be considered part of this syndrome (5, 75). A schematic representation of TDS is depicted in Figure 4.

Image testi-canc-pathogen_fig-4.jpg

Figure 4. Schematic illustration of aetiology and pathogenesis of disorders grouped within Testicular Dysgenesis Syndrome (TDS).

The TDS concept implicates disturbed function of testicular somatic cells (Leydig- and Sertoli cells) caused by inherited genetic mutations /polymorphisms in combination with environmental /lifestyle factors acting during early development. Dysfunction of the somatic cells results in disturbed hormonal homeostasis and causes impaired germ cell differentiation. Depending on the severity of the impairment, multiple outcomes or phenotypes may occur, ranging from reduced anogenital distance (AGD), genital malformations to testicular cancer. Note that the most severe forms of TDS (disorders of sex development with gonadoblastoma (GDB) or GCNIS are the least frequently seen, whereas the mildest forms, such as impaired spermatogenesis are quite common. Modified from Skakkebæk et al., Hum Reprod 2001 (3) and Physiol Rev 2016 (5).

Genetic Factors and Polymorphisms Predisposing to TGCT

Tumors derived from germ cells via GCNIS stage are characterized by the presence of a nearly universal aneuploidy and amplification of chromosome 12p, often in the form of an isochromosome (76). Strikingly few oncogenic mutations have been reported in TGCTs, and the short list of affected genes includes only KIT (predominantly in seminomas) and KRAS (77, 78, 79). Recent studies using next generation sequencing technology have identified other genomic aberrations in TGCT, including some recurring chromosomal gains or losses (in particular loss of chromosome Y), and fusion transcripts (79, 80).

Although testicular cancer in most cases is a sporadic disease, the familial risk of testicular GCT is among the highest when compared to other cancers; brothers and sons of TGCT cases have an 8-10-fold and 4-6-fold increased risk, respectively (81). A recent twin study from Nordic countries estimated the heritability of testicular cancer as 37%, with 24% attributed to shared environment (82).

Several genome-wide association studies (GWAS) have been performed in recent years, revealing a growing number of significant genetic associations with TGCT risk (83, 84, 85, 86, 87, 88). Some of the identified single nucleotide polymorphisms (SNP) are in coding regions, but most are in the introns or in close vicinity to coding sequences of genes, so a few can be directly implicated as associated with the pathogenesis of TGCT, and the rest are useful as genetic markers. The strongest association (identified in all GWAS), and most interesting from the biological point of view, is with a cluster of SNPs within or near KIT ligand gene (KITLG) as well as in some genes downstream of the KIT/KITLG/MAPK signaling pathway, e.g. SPRY4. The KIT ligand activates a tyrosine kinase receptor KIT and this pathway is essential for germ cell migration and survival, and is highly active in GCNIS and seminomas (27, 78, 89, 90, 91). Among other significant SNPs associated with TGCT risk, the most interesting were DMRT1, a transcription factors involved in sex differentiation and regulation of meiosis (92, 93), PRDM14 and DAZL, factors involved in primordial germ cell specification and differentiation, and several genes in the telomerase and DNA repair pathway (85, 86, 88). Two most recent meta-analytic GWAS studies performed by international consortia, identified numerous additional significantly associated loci, and raised the proportion of heritable cases of testicular cancer to nearly 40% (94, 95).

Current Views on the Etiology and Environmental Influences

In spite of significant recent inroads into the understanding of the pathogenesis of TGCT, etiology of these tumors, and especially the reason for the rise in incidence, remains obscure. The high incidence of testicular cancer in subjects with congenital errors of gonadal development and sexual differentiation strongly implicates the involvement of intrauterine factors and perinatal factors. We believe that the neoplastic transformation of male germ cells occurs during their pre-meiotic development, and this happens preferentially in individuals with genetic susceptibility. GCNIS cells and primordial germ cells share some distinct features, such as expression of embryonic pluripotency factors, low DNA methylation and constellation of miRNAs and histone modifications (4, 5, 15, 25, 26, 35, 37, 96, 97).

The mechanisms of neoplastic transformation of early germ cell are not known. There is a growing consensus that there may be multiple mechanisms and testicular cancer is a multifactorial and polygenic disorder. A disturbance in the fetal programming of gonadal development may be a result of an intrauterine hormonal imbalance, which in turn may be caused by a genetic disorder or by an impact of an exogenous factor targeting a key pathway, e.g. androgen signaling, KIT-KITLG signaling or a TGF-beta superfamily regulation (including Nodal pathway), leading to a delay in the testis development and maturation of fetal gonocytes (3, 4, 98).

As mentioned above, the rising incidence of testicular cancer in well-developed countries suggests a possible adverse influence of environmental or lifestyle-related causative factors. In recent decades a great number of potent natural and synthetic hormones and hormone antagonists have been identified in environment. Observations in wildlife and experiments in laboratory animals exposed to synthetic hormones and a broad range of endocrine disrupters suggest that these substances can cause a disturbance of hormonal milieu of the developing gonad and disturb differentiation of early germ cells (3, 4, 5, 99, 100). Whether or not the endocrine disrupters have contributed to the rise in testicular cancer remains to be demonstrated. Human exposure studies are difficult, mainly because of the long time interval between the vulnerable period of early development and manifestation of cancer development in adulthood. There is also the problem of the plethora of confounding factors, including mixtures of possible contaminating factors present in food, packaging, cosmetics and other products of daily life. Therefore, the evidence for the role of endocrine disrupters in the etiology of TGCT remains scarce. Among few existing data one can mention a higher serum levels of some persistent organic pollutants found in mothers of men with testicular cancer (101), and a greater burden of p,p′-DDE (a DDT metabolite) in serum of testicular cancer patients (102). Among other environmental risk factors for TGCT a heavy use of marijuana (cannabis) has been consistently reported (103, 104). For more information on the existing evidence and possible mechanisms the reader is referred to review articles on the subject (105, 106, 107). It is clear that further studies in large cohorts of patients and controls are needed to identify the causative factors behind the observed testicular cancer epidemics.

Diagnosis and Staging of Testicular Germ Cell Neoplasms: Testicular Biopsy

The preinvasive stage of GCNIS is asymptomatic, so diagnosis at this early stage is sporadic. It happens usually in cases with unilateral testicular atrophy, in the contralateral testis in patents with unilateral testicular tumors, or in individuals from high-risk groups, such as DSD or infertility. Surgical testicular biopsy is currently the only sure diagnostic procedure for GCNIS diagnosis. Even though the procedure does not completely eliminate the risk of second cancer, false-negative cases (approximately 0.03%) and post-biopsy complications are rare, (108, 109, 110, 111). Experience in the use of needle biopsies for GCNIS diagnosis is limited and this method is not commonly used in the clinic. However, examination for GCNIS by immunohistochemistry can be recommended in the leftover material after testicular sperm extraction in fertility clinics (112).

GCNIS is present in the testis contralateral to a testicular tumor in approximately 4% to 8% of cases (71, 113, 114, 109, 111). Our recommendation is, therefore, to perform a contralateral biopsy at the time of surgery for the primary unilateral testicular cancer in order to exclude the presence of GCNIS (and bilateral neoplasia), especially in men with small testes or a history of cryptorchidism. In a man older than 50, the biopsy can be omitted because of a minimal risk of GCNIS. In young men with normal size testicles, two-site biopsy may be considered to avoid a false-negative biopsy (114). Added benefits of a biopsy are a good assessment of the fertility potential of the other testicle, and the peace of mind for the patient concerning the possibility of bilateral cancer. However this issue remains controversial and the practice varies in different countries. Contralateral biopsies are routinely performed in all TGCT patients in Denmark and in some centers in the Netherlands, Germany, Austria, Switzerland and Sweden (109, 111). Most of other countries adopted a recommendation of the European Association of Urology (115) to take contralateral biopsies only in patients at the greater risk of GCNIS, identified by a history of cryptorchidism, a small testis volume (<12 ml) or testicular microlithiasis (73, 74, 109). In the multiethnic countries, such as the USA and Canada, where the prevalence of bilateral TGCT may be lower than in Northern Europe (69), the contralateral biopsy is rarely practiced.

Technical aspects of surgical biopsy are important and care has to be taken to avoid damage to the specimen. A small piece of tissue (approx. 3x3x3 mm) should be dropped directly to a container with a fixative solution (for morphology evaluation Bouin's- or Stieve's- solution is preferred, because formalin causes shrinkage artefacts, while for immunohistochemistry buffered formalin is preferred). The histological examination of the biopsy, should be aided by immunohistochemical staining for one of the known marker proteins e.g. placental-like alkaline phosphatase (PLAP), PDPN/M2A/D2-40, OCT-3/4 or AP2γ (24, 108, 109, 110, 111, 116). An example of immunohistochemical PLAP staining for GCNIS detection is shown in Figure 1).

Semen Analysis

Semen analysis cannot as yet be used alone for detection of early stages of testicular cancer. Poor spermatogenesis is a good indication that the patient may be in risk of harboring GCNIS (67, 68, 73, 74, 117). Moreover, it has been known for a long time that GCNIS cells may be occasionally found in semen (118). Over the years, progress has been made towards establishing better quality immunocytochemical detection of GCNIS cells in semen, and an improved automated double-staining-based assay has been developed (119, 120, 121). Further improvement of sensitivity is needed, but this non-invasive approach may - if successful - eliminate the need for the contralateral biopsy in the future.

Scrotal Imaging (Ultrasonography)

Scrotal ultrasonography has been increasingly popular for assessment of the testicles and should be performed routinely if there is a suspicion of testicular malignancy, even if there is no history of cryptorchidism or fertility problems. Testicles afflicted with GCNIS frequently have an irregular echo pattern that may be associated with the presence of testicular microliths or hyaline bodies (122, 123, 124). However, ultrasonic microlithiasis is not always confirmed histologically, on the other hand, microliths are one of the signs of dysgenetic testes and may be seen in testicular atrophy or cryptorchidism, without GCNIS (122). Despite the problem of lack of specificity, a sonographic finding of testicular microlithiasis and an irregular echo patter should alert the clinician to consider malignancy and refer the patient for a surgical biopsy if other indications are present; such as testicular atrophy, poor semen quality or a history of cryptorchidism (115, 122, 123, 124).

In addition to microlithiasis, ultrasonographic examination will detect small not yet palpable tumors, and help in differential diagnosis of other conditions, e.g. orchitis, epididymitis, hydrocoele and varicocoele. Sensitivity of the ultrasound to distinguish between small tumors and other incidental lesions can be improved by supplementary imaging techniques, e.g. strain ratio elastography, which allows assessment of tissue elasticity in real time (125). Other imaging methods, such as MRI (magnetic resonance) remain rarely used for diagnosis of TGCT, mainly because of a limited experience and high cost of the procedure. However, scrotal MRI and elastography are considered helpful if there is a discrepancy between the ultrasound image and clinical history, so the patient can be spared orchiectomy in benign lesions (125, 126, 127).

Serum Tumor Markers, Detection and Monitoring of Overt Tumors

In the vast majority of cases a scrotal mass is usually the first presentation of testicular cancer, with tenderness reported by only few patients. In a few percent of testicular cancer cases, the presenting symptoms are the result of metastatic disease. They are usually uncharacteristic and may include lumbar pain, palpable abdominal mass, supra-clavicular lymph node enlargement and in rare cases pulmonary symptoms (1).

The majority of primary and metastatic germ cell tumors secrete protein products that can be detected in circulating blood. These biochemical serum tumor markers are very helpful in diagnosis and monitoring of testicular cancer (128, 129). Among nonseminomatous tumours, choriocarcinoma, which resembles gestational trophoblast, produces large quantities of human chorionic gonadotropin (HCG) and yolk sac tumor, which is similar in morphology to the embryonic yolk sac, secretes alpha-fetoprotein (AFP) (24, 130). In addition, lactate dehydrogenase (LDH) may be secreted by both nonseminoma and seminoma. LDH levels in serum tend to be higher in patients harboring tumors with an increased copy number of chromosome 12p, consistent with the genomic location of the LDHB gene (131). Increased concentration of LDH in the absence of AFP and HCG suggests the presence of seminoma. The most important in clinical practice are HCG and AFP, since they are very sensitive markers for nonseminomas, which in many cases have a more malignant clinical course than seminoma (1, 129, 130). It is important to keep in mind that TGCT rarely occur in pure histological forms, and for example HCG may be sometimes detected in serum of patients with seminoma due to the presence of some giant cells in a tumor mass. In preinvasive GCNIS and in most cases of pure classical seminoma, none of the above mentioned markers are detectable in serum. Other markers, e.g. PLAP, have been adapted for use as serum assays, but these have not been used in clinical practice (132).

Promising emerging serum markers are micro-RNAs (miRs), among which miR 371-3, and miR 367 have already proven to be valuable for detection of TGCTs in seminomas and nonseminomas, except for differentiated pure teratomas (133, 134, 135, 136). These specific miRs types are already produced in GCNIS cells (35), so ongoing research projects are striving to develop a miR-based assay to detect GCNIS, but the sensitivity is still too low to apply this method in the clinic (137).

The measurements of circulating tumor markers help in prognosis. There is a tendency for higher levels of tumor markers to be associated with a poorer prognosis. However, a careful staging is necessary in each case to decide for the most appropriate treatment strategy (1, 115, 128). In addition to serum markers, other procedures help to evaluate the spread of disease, such as bipedal lymphangiography, X-ray of the thorax, retroperitoneal CT scan or ultrasonography (1, 138, 139). Histopathological evaluation of the orchidectomy specimen is also a part of the staging, and the important risk factors, tumor size, vascular/lymphatic or rete testis invasion, and the presence of pure embryonal carcinoma (140, 141). These procedures help to classify patients into one of the staging systems.

The most commonly used staging and prognostic classification system is the TNM (tumor, node, metastases) System of the International Germ Cell Cancer Collaborative Group (GCCCG) and the American Joint Committee on Cancer (AJCC) (142). The stage grouping recently updated by WHO

(2), is shown in Table 2.

Table 2Stage grouping according to TNM classification (adapted from 2, 129)

Stage 0 (pTis): Germ cell neoplasia in situ
Stage IA (pT1, N0, M0, S0): Tumor limited to testis and epididymis
Stage IB (pT2-4): as IA but with vascular/lymphatic, tunica or scrotal invasion
Stage II (any pT, N1-3, M0, S0-1): Metastasis in lymph nodes, serum markers normal or moderately increased
IIA (N1): lymph nodes <2 cm
IIB (N2, S1): lymph nodes >2 cm but <5 cm
IIC (N3, S1): lymph nodes >5 cm
Stage III (any pT, any N, M1, S0-3): Distant metastasis (spread beyond regional nodes)
IIIA (M1a, S0-1): Spread to non-regional nodes or lung
IIIB (M1a, S2): as IIIA but high serum markers
IIIC (M1a-b, S3): distant metastasis to sites other than IIIA, or very high serum markers

Footnote: pT=primary tumor, N=regional lymph nodes, M=distant metastasis, S=serum tumor markers (X=unknown, not available)

Management of GCNIS and Testicular Germ Cell Cancer

Early diagnosis of testicular neoplasia at the stage of GCNIS, followed by adequate treatment of GCNIS is capable of preventing progression to invasive tumors. Unfortunately, the vast majority of cases progress unnoticed to overt tumors. However, germ cell tumors are extremely radio- and chemo-sensitive and have apparently very high propensity to apoptosis, likely mediated by p53 (143, 144). Because of this sensitivity, and thanks to cisplatin-based chemotherapy regimens, TGCT is a highly curable malignancy, with more than 80% of patients reaching a sustained complete remission (145).

We give here only very general information concerning management of testicular cancer. The reader should consult specialized oncologic and urologic literature for management options pertinent to treatment-resistant tumors and metastatic disease.

Treatment of GCNIS

The following management options for GCNIS are available depending on a specific situation (146):

  • Orchiectomy - is the curative treatment with the highest assured success rate. It should be always performed on a testis with GCNIS or localized tumor when the second testis is not affected by testicular neoplasia (including GCNIS).
  • Radiotherapy - low dose radiotherapy is a good alternative to orchiectomy in some cases, for example when GCNIS is present in the contralateral testis, so the patient can be spared a total castration and lifelong androgen replacement therapy. The efficacy of radiotherapy with doses as low as 16 Gy was demonstrated in the early studies (147). Even though the lower dose better preserves the function of Leydig cells, more recent studies and current EAU guidelines recommend a dose of 20 Gy, given in fractions of 2Gy (115, 148). This dose of radiation will almost always destroy also normal germ cells, so radiotherapy may be delayed in patients who wish to secure natural conception of a child.
  • Chemotherapy - is not an option to treat GCNIS, because a persistence or relapse has been reported in a high proportion of patients (149, 150). Furthermore, in some cases of extragonadal germ cell tumors treated with chemotherapy, testicular GCNIS progressed to metachronous overt testicular tumors (151). However, if a patient with disseminated disease receives a chemotherapy, he will have a lower risk of metachronous bilateral TGCT.
  • Surveillance - is potentially hazardous since GCNIS may progress to invasive cancer at any time, but may be an option after careful informed discussion of risks and monitoring with ultrasound examinations, especially if the patient wishes to defer treatment temporarily for the purpose of paternity.

In cases of desired fatherhood, and in all cases of very young men, semen analysis and cryopreservation of semen samples, if viable spermatozoa are detected, should be done.

Treatment of Overt Seminomas and Nonseminomas

As far as the treatment of invasive germ cell tumors is concerned, the reader should consult the specialized urology and oncology guidelines. Radical orchiectomy remains the primary treatment method of choice. The patients ought to undergo a CT or MRI scan of the abdomen, thorax and pelvis to look for possible spread of the disease (152). Primary dissection of retroperitoneal lymph nodes (RPLND), previously commonly practiced, especially in USA, is currently performed sparingly, only in selected patients, preferably by nerve-sparing techniques (1, 152, 153). Adjuvant radiotherapy, e.g. of the retroperitoneal/para-aortic field, which was previously routinely used, is no longer recommended, because of the long-term risk of secondary malignancies (1, 115, 152, 153).

Prior to surgery, patients with TGCT should be offered andrological consultation. Semen analysis and cryopreservation of spermatozoa should be offered to all patients. It is also advisable to perform an andrological /endocrinological evaluation of the patient before surgery, including serum testosterone, gonadotropins and inhibin B, if possible. In patients with bilateral tumors or only one testicle, it is important to perform pre-operative semen analysis. In the patients who have azoospermia or cryptozoospermia, and in whom it was impossible to retrieve sperm pre-operatively, testicular sperm extraction (TESE) at the time of orchidectomy (‘onco-TESE’) may be attempted in specialized centers (154). The best predicting factor of sperm retrieval is a small size of the tumor, because a chance of finding tubules with ongoing spermatogenesis increases with the distance from the tumor (155). After the surgery, careful staging must be performed, based on the histopathologic evaluation of the tumor (141) and serum markers (see section ‘Serum tumor markers’ above).

Methods of post-surgical management of overt TGCT are variable, depending upon the histological type of tumor (seminoma vs nonseminoma), levels of serum markers and stage of disease and the presence of residual retroperitoneal masses (1, 115, 138, 139).

Pure seminomas have a good prognosis and 88% of patients with stage I seminoma (tumor confined to the testis) do not require any treatment after the surgery, thus most of the centers practice a surveillance strategy (138, 153). Nonseminomas have a somewhat poorer prognosis (relapse rate is around 30%) and in some centers these are treated more aggressively, including one course of adjuvant chemotherapy (156), but most centers recommend a surveillance strategy (1, 140, 153, 157). Histological seminomas with the presence of serum markers (HCG>200 IU) should be treated as nonseminomas (141, 139). If serum markers are elevated, an oncologist must be contacted immediately to coordinate management. In the vast majority of cases the surgery should be performed first, unless there are signs of a life-threatening metastatic disease (139).

The most common post-surgical management of disseminated tumors is systemic combination chemotherapy with a combination of cytotoxic drugs, such as cisplatin, etoposide, bleomycin, vinblastine and methotrexate, in selected cases associated with adjuvant radiotherapy, which should be used sparingly, because of a higher risk of adverse late-effects (1, 115, 145, 138, 139, 152). The standard first line chemotherapy regimen is BEP (bleomycin, etoposide and cisplatin), administered in 3 or 4 cycles, depending on the patient’s prognosis. It is very important to make a dynamic assessment of the progress of treatment through the early stages of chemotherapy, thus monitoring of serum markers is obligatory (128). In patients with poor prognosis or resistance to chemotherapy, salvage regimens and complex surgery for residual tumors need to be performed. Overall the management is difficult, thus it should be carried out in specialised tertiary centres (1, 152).

The majority of relapses occur within 2 years of the initial treatment but late relapses are observed in some cases, especially in nonseminomas, therefore individual management of each patient and lifetime follow-up is advocated (1, 152, 156, 157).

Andrological follow-up should be coordinated with the post-surgery oncological management and controls (see the last section on ‘Endocrine problems and late effects of testicular neoplasms’).

SEX CORD-STROMAL TUMORS OF THE TESTIS

In adults, sex cord-stromal tumors of the testis are found in less than 5 % of all testicular tumors, whereas in children, these tumors are found in up to 40 % of cases (158, 159, 160, 161, 162). Most of these tumors are benign, only around 5% have malignant characteristics (2). The recently updated classification of the tumors by WHO (2016) is shown in a simplified form in Table 3 (2, 146).

Sex cord-stromal tumors are derived from somatic cells; Leydig cells, and Sertoli/granulosa cells. Despite a different cell of origin, some stromal tumors are sometimes misinterpreted as seminoma. A number of features can be used to distinguish sex cord-stromal tumors from germ cell tumors; Inhibin A and B are the best serum markers for this purpose (163, 164, 165). Inhibin A appears to be a common immunohistochemical marker for sex cord-stromal tumors, including Leydig cell, Sertoli cell and juvenile granulosa cell tumors (163, 166). Sertoli cell tumors are positive for anti-Mullerian hormone (167) and GATA-4 (168), while Leydig cell tumors express steroidogenic enzymes and INSL3 (169). Other useful immunohistochemical markers, include SOX9, calretinin, CD99, SF1 (2).

Table 3

Sex cord stromal tumors of the testis (adapted from ref. 2)

  • Leydig cell tumors (benign or malignant)
  • Sertoli cell tumors (SCT)
    - Malignant SCT
    - Large cell calcifying SCT
    - Intratubular large cell calcifying SCT
· Granulosa cell tumors
- Juvenile-type granulosa cell tumors
- Adult-type granulosa cell tumors
  • The fibroma-thecoma tumors
  • Mixed sex cord-stromal tumors
  • Unclassified sex cord-stromal tumors

Leydig Cell Hyperplasia and Tumors

Leydig cells are located in the interstitial compartment of the testis and are involved in the development of secondary male characteristics and maintenance of spermatogenesis. Although Leydig cells in adult men are considered to be a terminally differentiated and mitotically quiescent cell type, in various disorders of testicular function, focal or diffuse Leydig cell hyperplasia is very common. Micronodules of Leydig cells are frequently seen in certain conditions associated with severe decrease of spermatogenesis or germinal aplasia, such as the Sertoli-cell-only syndrome (Del Castillo syndrome), cryptorchidism, or Klinefelter's syndrome (170, 171). A term 'Leydig cell adenoma' is used when the size of a nodule exceeds several- fold the diameter of a seminiferous tubule. It is unknown whether Leydig cell adenomas can progress further to form overt Leydig cell tumors, but even if it were the case, it is exceedingly rare. Morphological heterogeneity of hyperplastic Leydig cells is noticeable in some cases, and it has been shown that the micronodules contain a large proportion of immature Leydig cells (170, 171).

The mechanism of Leydig cell hyperplasia in the human male is still poorly understood. The disruption of hypothalamo-pituitary-testicular axis leading to an excessive stimulation of Leydig cells by LH can play a central role (170). This in turn leads to an increased renewal of immature, adult-type Leydig cells from their precursors. The immature cells are characterized by low numbers of Reinke cristals, a relatively high expression of a mesenchymal factor DLK1 and low amounts of INSL3 (171, 172, 173). However, Leydig cell hyperplasia is distinct from tumors that are usually solitary. Leydig cell hyperplasia and adenomas can be easily induced in rodents by administration of estrogens, gonadotropins and a wide range of chemical compounds. Whether or not humans would be similarly susceptible to environmental effects remains to be elucidated.

Leydig cell tumors account for one to three percent of testicular neoplasms and occur in all age groups (for reviews, see 2, 162, 174). Approximately 20 % are found before the age of 10, most often between five and ten years of age. In a subset of cases of Leydig cell hyperplasia or tumors, activating mutations of the LH receptor (175, 176, 177) or G proteins (178, 179) can be detected. Constitutively activating mutations of LH receptor cause early Leydig cell hyperplasia and precocious puberty (175, 177, 180). Similarly, constitutively activating mutations of Gs-protein in Leydig cells or inactivation of PKAR1A (protein kinase cyclic adenosine monophosphate-dependent regulatory type 1 alpha) lead into hyperplasia and endocrine hyperactivity (162, 179). In adult Leydig cell tumors, germline fumarate hydratase mutations have been identified in a few cases which also had hereditary leiomyomatosis and renal cell cancer (181).

Precocious puberty (so-called testotoxicosis) is the presenting symptom in most of cases of Leydig cell adenomas or tumors in children, due to the excessive production of androgens, mainly testosterone that cause growth of penis, pubic hair, accelerated skeletal and muscle growth, advancement of bone age, skin changes (acne, comedos, hair greasing) and adult-type odor of sweat. Androgen secretion in the pediatric cases is gonadotropin independent, and therefore LH and FSH remain low in spite of external signs of puberty (177). Approximately 10 % of the boys also have gynecomastia that is caused by estrogens produced in excess due to aromatase activity in some of the tumors or peripheral aromatization of testosterone. In adults, gynecomastia is found in approximately 30 % of patients (182). The excessive androgen secretion rarely causes notable effects in adults. However, gynecomastia is sometimes associated with loss of libido, impotence, and infertility. The fertility problems are usually due to impairment of spermatogenesis, caused by suppression of gonadotrophins by excessive testosterone or estradiol production. Removal of a steroid-producing tumor would often lead to recovery of spermatogenesis with time. Malignant somatic tumors are hormonally active only in exceptional cases (2, 162).

Leydig cell tumors are always benign in children and can be treated with surgical enucleation when the tumor is encapsulated (160). In adults malignant Leydig cell tumors have been found in 10-15 % of patients, and inguinal orchidectomy is often used (183), while testis-sparing enucleation remains an option also in adults (159). The presence of cytologic atypia, necrosis, angiolymphatic invasion, increased mitotic activity, atypical mitotic figures, infiltrative margins, extension beyond testicular parenchyma, and DNA aneuploidy are associated with metastatic behavior in Leydig cell tumors (162, 182, 184) (see Figure 5).

Image testi-canc-pathogen_fig-5.jpg

Figure 5. Histology of a Leydig cell tumor.

The appearance of tumor cells resembles normal Leydig cells. A section stained with hematoxyllin-eosin (HE).

Malignant tumors have not responded favorably to conventional chemotherapy and irradiation (183). Survival time has ranged from 2 months to 17 years (median, 2 years), and metastases have been detected as late as nine years after the diagnosis (162, 182). Therefore follow-up of patients with malignant Leydig cell tumors has to be life-long. The remaining testis may be irreversibly damaged by longstanding high estrogen levels, resulting in both permanent infertility and hypoandrogenism (182, 184).

Testicular Adrenal Rest Tumors

Excessive secretion of adrenocorticotropin (ACTH) in poorly controlled 21-hydroxylase deficiency (congenital adrenal hyperplasia, CAH) or Nelson syndrome (postadrenalectomy status) may lead to development of hyperplastic interstitial nodules called adrenal rests in the testis resembling Leydig cell tumor or hyperplasia (174, 185, 186). These cells are hormonally active in secreting androgens. It is important to remember that the adrenal rests are almost invariably bilateral, whereas the Leydig cell tumors are usually unilateral. Adrenal rests can be effectively treated by appropriate glucocorticoid substitution of the patient, which leads to gradual regression of the 'tumor' in 75 percent of cases (185, 186). Adrenal rest tumors have to be distinguished from benign hyperplasia, adenomas and malignant Leydig cell tumors (169). Histopathological and endocrine evaluation covering both testicular and adrenal steroids and pituitary gonadotropins and ACTH are important to make differential diagnosis (169, 185). Obviously it would be an error to orchidectomize the CAH patients with adrenal rest tumors, since the tumors are always benign and only some of them continue to be active after appropriate glucocorticoid substitution. In these rare cases, testis sparing surgery could be considered.

Sertoli Cell Tumors

Sertoli cells are the somatic cells in the seminiferous epithelium giving structural, metabolic and hormonal support to spermatogenic cells. Sertoli cells terminally differentiate and cease their proliferation at puberty. In rare infantile cases, multiple foci of proliferating Sertoli cells have been described and proposed to be early intratubular forms of Sertoli cell tumors (187). The classification of these tumors was revised in 2016 by WHO (2, 162), as shown in Table 3.

Sertoli cell tumors which do not belong to a syndrome are called ‘not otherwise specified’ (NOS), and about 5% of these tumors are malignant and can metastasise. The tumors typically are composed of sex cord cells with tubular differentiation, with a subset of tumors hyalinized, previously classified as sclerosing variant (2). Some tumors often contain lipid droplets but do not show any endocrine activity. The molecular origin is known only in a small proportion of tumors with sclerosing appearance, in which CTNNB1 mutations causing nuclear accumulation of β-catenin were identified (188).

The age of the patients with Sertoli cell tumors, ranges from 18 to 80 years, but most of them are young adults (median age 30). Out of 60 patients, only four were younger than 20 years old in the series reviewed by Young et al. (189). The tumors occurred in descended testes and were always unilateral. An infiltrative margin was found in four cases, but most of the tumors were well demarcated. The tumors were hormonally inactive, and only two patients with alcoholic cirrhosis also had gynecomastia. Eighteen pediatric cases were reported from the Kiel Pediatric Tumor Registry (159), but perhaps the histopathologic pattern was somewhat different, because the age of the children was very young, ranging from 0 to 14 months (median 4 months). Juvenile Sertoli cell tumors often showed infiltrative growth into adjacent tissue, dense cellularity and considerable proliferative activity. However, after surgical excision no local recurrences and no metastases occurred. Thus, these patients have a good prognosis. Sertoli cell tumors can be treated by orchidectomy, and retroperitoneal lymphadenectomy is indicated only when there is radiographically detected retroperitoneal involvement (190).

Large-cell calcifying Sertoli cell tumors, are frequently found in association with two distinct multiple neoplasm syndromes, Carney complex and Peutz-Jeghers syndrome, however in the latter syndrome, the tumors belong to a distinct ‘intratubular’ morphological group (2, 162, 191).

Carney complex is characterized by skin myxomas, heart myxomas, typical skin pigmentations, adrenal and testicular tumors, but other tumors can also occur (192). The testicular tumors are large-cell calcifying Sertoli cell tumors that are multifocal and bilateral, and should be distinguished from teratomas (Figure 6) (162, 193, 194). The tumors appear usually during the second decade of life (195). Only one malignant case has been reported in association with Carney complex (in an adult patient), whereas seven malignant tumors were reported in other patients with large-cell calcifying Sertoli cell tumors (195). These patients were older than 25 years. The malignant cases were unilateral and solitary in contrast to bilateral and multifocal occurrence of testicular tumors in Carney complex. Large-cell calcifying Sertoli cell tumors are usually not hormonally active, although elevated levels of serum inhibin B or testosterone have been reported, but other tumors of Carney complex, including Leydig cell tumors, can cause endocrine manifestations (165, 190, 191).

Image testi-canc-pathogen_fig-6.jpg

Figure 6. Large cell calcifying Sertoli cell tumor isolated from a 12-year-old boy.

The neoplastic tubules contain only large pale Sertoli cells and visible calcifications in the lumen (stained with PAS). Adjacent normal tubules show advanced spermatogenesis.

Two genetic loci for Carney complex have been identified on chromosome 2p16 (196) and 17q23-24 (197). The genetic loci are different from that in Peutz-Jeghers syndrome (198). Germline mutations identified in Carney complex most often occur in type I-alpha regulatory subunit of protein kinase A, PRKAR1A (199, 200). Inactivating mutations of phosphodiesterase 11A212 and phosphodiesterase 8B213 are associated with bilateral adrenocortical hyperplasia in Carney complex patients. Genetic variation of the phosphodiesterase 11A gene can modify the development of the testicular tumors (191, 201). Thus, molecular genetic diagnosis is now available to many of these patients. Association of large-cell calcifying Sertoli cell tumors with other neoplasms, particularly heart myxomas in Carney complex and gastrointestinal tumors in Peutz-Jeghers syndrome, should be kept in mind to reach an early diagnosis of these potentially fatal diseases.

Sertoli cell tumors in Peutz-Jeghers syndrome are similar in appearance to those found in Carney complex patients, but can be distinguished by typical intratubular proliferation of lightly eosinophilic cells with prominent basement membrane deposits, and occasionally, features of ovarian sex-cord tumors with annular tubules (162). These tumors may have strong aromatase activity and therefore be associated with gynecomastia (202). No malignant testicular tumors have been reported in Peutz-Jeghers patients, but they have a highly increased risk of other neoplasms, especially colorectal, breast, pancreatic and ovarian cancers (191). Germline loss-of-function mutations in the STK11/LKB1 gene that encodes for a serine-threonine kinase causes Peutz-Jeghers syndrome in the majority of patients, allowing molecular genetic diagnostics (191, 202, 203).

Sertoli cell tumors of Carney complex patients should be treated conservatively in children to give them a possibility for sperm banking before orchiectomy comes necessary (204). If precocious puberty and/or gynecomastia appear, aromatase inhibitors and antiandrogens can be used to prevent estrogen formation and androgen action (196, 204).

Granulosa Cell Tumors

Juvenile-type granulosa cell tumors are the most common somatic testicular tumors in infants and occur during the first 6 months after birth (2, 159, 162, 205). Prominent differentiation into follicles and immature nuclei distinguishes juvenile granulosa cell tumors from other Sertoli cell tumors that express tubular differentiation (162, 206). Most of the immunohistochemical markers in these tumor types are similar, e.g., inhibin, calretinin, but also distinct, e.g. expression of FOXL2 (206). Juvenile granulosa cell tumors always have a good prognosis. Juvenile granulosa cell tumors have been found in undescended testes with abnormal sex chromosomes and ambiguous genitalia (for references see 159). Testicular tumors do not show endocrine hyperactivity, in contrast to ovarian juvenile granulosa cell tumors. Aberrant WNT signaling (207) and stimulatory G-protein mediated signaling (208) have been linked with juvenile granulosa cell tumors.

Adult-type granulosa cell tumors are comparable to the ovarian tumors, but are extremely rare (2, 162, 209). These tumors occur in adults at an average age of 42 years. Twenty percent of the patients have shown gynecomastia due to the hormonal activity of the tumor. Most of the tumors are benign, but some malignant cases have also been reported (2). Mutations in FOXL2 have been reported in adult ovarian granulosa cell tumors but only few secondary mutations have been found in testicular granulosa cell tumors (210, 211).

Fibroma-Thecoma Tumors

These exceedingly rare neoplasms are composed of fibroblastic cells of testicular stroma or tunica albuginea (2). These tumors are reported to be benign.

Mixed and Unclassified Sex Cord-Stromal Tumors

Tumors consisting of more than one stromal or tubular component or have indeterminate morphology are classified as mixed and unclassified sex cord-stromal tumors (2, 162).

Sex cord-stromal tumors can contain combinations of Leydig, Sertoli, granulosa, and theca cells, and are therefore called mixed tumors (162). Leydig cells can be difficult to recognize in these tumors. These tumors are rare and can occur at any age. Depending on the predominant cell type the tumors may behave differently. Gynecomastia, as a sign of endocrine activity, can be found in ten percent of patients (2). These tumors are always benign in children, but in adults malignancy can be found (208). Thus, most of the patients can be treated by orchidectomy, and lymph node dissection is indicated only in cases with overt malignant features on microscopic examination.

OTHER TUMORS

Other tumors occurring in the testis are divided into miscellaneous and hematolymphoid tumors (2, 162). The first group includes ovarian epithelial-type tumors, serous or mucinous cystadenomas, adenocarcinomas, Brenner tumor, xantogranuloma and hemangioma. The hematolymphoid tumors comprise malignant lymphomas (B-cell, NK/T-cell or follicular), plasmacytoma, myeloid sarcoma and Rosai-Dorfman disease (2, 162). In addition, testicular spread of malignant acute leukemia is common in young boys, and metastases from other solid tumors including the prostate gland, colon, kidney, stomach, pancreas, and malignant melanoma can be found in the testis of adults.

ENDOCRINE PROBLEMS AND LATE EFFECTS OF TESTICULAR NEOPLASMS

Relative imbalance of androgen signaling (excess or deficiency) causes the most pronounced secondary endocrine symptoms associated with testicular tumors. Testosterone is produced by tumors, such as Leydig cell tumors, or by normal Leydig cells stimulated by large amounts of hCG from some germ cell tumors. Excess of androgens would lead to precocious puberty in children (175, 176, 177. In addition, aromatisation of androgens leads to a relative excess of estrogens, which causes impairment of spermatogenesis in adults and gynecomastia at any age (202, 212).

Testicular dysfunction in young adult patients with testicular cancer, who usually are in their best reproductive age, is a serious clinical problem. Patients with testicular tumors have poor spermatogenesis and decreased fertility even before the overt tumor has developed (66, 67, 68, 117) and before cytotoxic treatment (213). Features include oligozoospermia, elevated LH levels, and a variable degree of testicular dysgenesis or atrophy in the biopsy, in some cases further complicated by the presence of GCNIS. Examination of a contralateral biopsy in a patient with a unilateral tumor may show a similar picture; with at least 5% risk of presence of GCNIS cells (71).

Testicular function is further disturbed by treatment of neoplasm. In recent years there is growing concern about adverse late effects of irradiation and chemotherapy (1, 152, 153, 214, 215). Refinement of the dosage must be considered in each patient individually, to eradicate the neoplasm with least possible damage to the endocrine function. The eradication of GCNIS or a tumor by irradiation in bilateral cancer cases leads also invariably to the disappearance of all germ cells and sterility. This underlines the importance of semen analysis and cryopreservation before treatment which will allow assisted reproduction treatment, if needed (216, 217, 218). It is also important to perform semen analysis in all patients interested in fertility before the treatment. If semen banking or pre-operative sperm retrieval is not possible or if the patient has azoospermia or cryptozoospermia, testicular sperm extraction (TESE) at the time of orchidectomy (‘onco-TESE’) may be the only chance of fertility, and can be attempted in specialized centers (154, 155). TESE and subsequent intracytoplasmic sperm injection (ICSI) may be also an option for fertility treatment in some cases of post-chemotherapy azoospermia (219). Additional problem in survivors of disseminated/advanced TGCT treated with radiotherapy or chemotherapy regimens is the increased risks of second cancers, cardiovascular disease, peripheral neuropathy, hepatoxicity and ototoxicity (214, 215, 220).

All patients treated for testicular cancer require careful assessment of their reproductive hormones and spermatogenic capacity with respect to their future fertility, sexual potency, possible need for androgen replacement therapy and general well-being. In long-term survivors fertility is impaired and signs of Leydig dysfunction manifested as low testosterone and high LH levels are common (221, 222, 223). A close follow-up by an endocrinologist /andrologist is important not only because of the reproductive system issues, but also because hypogonadism is a major risk factor for metabolic syndrome later in life (223, 224). Of importance are quality of life issues related to prolonged anxiety and stress, loss in socioeconomic status or unemployment, so a psychologist/social advisor should be added to the multidisciplinary team of experts caring for survivors of TGCT (152, 225, 226).

ACKNOWLEDGEMENTS

The authors thank the research and clinical teams at their departments for the contribution to the studied summarized in this review. The authors are also grateful to Prof. R. McLachlan for the critical review of the chapter. The work was supported by grants from numerous foundations, with the biggest contributions from the Danish Cancer Society, the Lundbeck Foundation, the Svend Andersen Foundation, the Danish Advanced Technology Foundation, Sigrid Juselius Foundation and Novo Nordisk Foundation.

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