Testis

Question 1

Cryptorchidism

Answer 1

• Absence of one or both testes in the scrotum
• Most common congenital abnormality of the genitourinary tract
• Associated with infertility and subfertility, testicular germ cell tumor, testicular torsion and inguinal hernia
• Seminoma is most common malignancy
• More common in preterm (30%) and low birth weight infants
• Concordance in sibling studies
• Associated with and increased risk in certain chromosomal abnormalities and genetic syndromes, such as Klinefelter and Prader-Willi syndrome
• 80% of undescended testicles palpated within inguinal canal or high scrotal area
• 20% of undescended testicles not palpated
• 50% lie in abdomen, 50% are atrophic

Micro:
* Histopathologic differences become much more pronounced after 2 years of life and are increased with delayed orchiopexy
* Peritubular fibrosis
* Seminiferous tubule atrophy
* Decreased / absent spermatogenesis
* Sertoli cell only seminiferous tubules: tubules with only bland, monotonous pale cells with granular cytoplasm attached to the basement membrane; absent germ cells and no spermatogenesis
* Sertoli cell nodule: nodules composed of immature elongated Sertoli cells (Pick adenoma) may be present; can have central microliths
* Increased microliths within seminiferous tubules
* Retained Leydig cells give appearance of hyperplasia
* Evaluate for germ cell neoplasia in situ (GCNIS): large, pleomorphic, basally located cells within the seminiferous tubules may be present; use immunostains for confirmation

Question 2

Hydrocele

Answer 2

• Accumulation of serous fluid between visceral and parietal layers of tunica vaginalis
• Idiopathic or associated with nonneoplastic or neoplastic lesions
• Note: thorough macroscopic examination and extensive sampling are needed to rule out mesothelioma
• Association with inguinal hernia, scrotal trauma, inflammation (epididymoorchitis) or tumors of the testis / paratestis
• Filarial hydrocele –> Late and chronic manifestation of filariasis, Due to parasite induced blockage and dysfunction of the lymphatic vessels

Micro:
* Loose connective tissue lined by a single layer of cuboidal or flattened mesothelial cells
* Lining may show mesothelial hyperplasia (both solid and papillary), squamous metaplasia or prominent atypia
* Usually clear luminal fluid
* Fibrinous exudate, chronic inflammatory infiltrate and fibrosis in longstanding cases, due to infection or hemorrhage
* Occasional presence of florid nodular collections of histiocytes and aggregates of incidental benign small blue cells of possible rete epithelial origin

Question 3

Orchitis

Answer 3

Three Types:
* Viral
* Bacterial
* Granulomatous

Viral Orchitis
* Most common cause —>Mumps
* Coxsackievirus B also relatively common
* Manifests with testicular pain
* Appears shortly after or during viral syndrome –> includes parotitis
* May be bilateral
* Acute inflammation
* Long term –> patchy interstitial fibrosis and atrophy of seminiferous tubules

Bacterial Orchitis
* Most common cause —> E.coli
* Acute or chronic
* Usually associated with infection elsewhere in the GU tract
* Often associated with bacterial epididymitis
* Prominent neutrophilic infiltrate with abscess formation
* Chronic bacterial orchitis may show granulomatous inflammation
* Lacks intatubular giant cells

Granulomatous Orchitis
* Usually a chronic process
* Associated with a variety of organisms
* Associated with systemic or extra testicular infection
* May be idiopathic
* Intratubular, non-necrotising granulomas are seen

Question 4

Testicular Torsion

Answer 4

• Twisting of the spermatic cord and its contents
• Surgical emergency
• Haemorrhagic infarction of the testis
• Twisting of the testicle along the spermatic cord results in reduced venous outflow and vascular congestion
• Prolonged twisting impairs arterial flow and ischemia
• Prolonged ischemia can result in necrosis

Macro:
* Depends on the degree of torsion and the time lapse since onset of symptoms
* Dark red testis with smooth external surface and solid consistency

Micro:
* Edema, congestion and interstitial hemorrhage with venous outflow obstruction
* Early conservation of germ cells, which later disappear
* Initial phase (< 6 hours): dilatation of seminiferous tubules with sloughing of germ cells and vacuolization of Sertoli cells
* Followed by pronounced hemorrhage and sloughing of all germ cells
* Necrosis of the seminiferous epithelium
* Hemorrhagic infarction of testicle

Question 5

Testicular Germ Cell Tumours

Answer 5

• WHO 2016
• GCNIS –> isochromosome 12 (i12p)

GCNIS-related TGCTs
* Seminomas
* Non-seminomatous mixed germ cell tumours:
Embryonal carcinoma
Yolk Sac tumours, post-pubertal type
Teratomas, post-pubertal type
Choriocarcinomas and other trophoblastic tumours
* Burned-out (regressed) germ cell tumours

Non-GCNIS-related TGCTs
* Yolk-sac tumours, prepubertal type
* Teratomas, prepubertal type
Dermoid cysts
Epidermoid cysts
Well-differentited tumours
Neuroendocrine tumours
* Spermatocytic tumors

Question 6

Germ cell neoplasia in situ (GCNIS)

Answer 6

• Common precursor of type II germ cell tumors (i.e., seminomas and most postpubertal nonseminomatous germ cell tumors of the testis)
• Associated with:
• uncorrected cryptorchidism
• ambiguous genitalia
• infertility
• prior history of contralateral postpubertal germ cell tumor or GCNIS

Micro:
* Neoplastic cells located along the basement membrane of seminiferous tubules (spermatogonial niche)
* Cells are large atypical gonocyte-like with abundant clear cytoplasm and large (10 - 11 μm) hyperchromatic nuclei with coarse chromatin, angulated borders and prominent nucleoli; these cells are similar in appearance as seminoma
* Affected seminiferous tubules frequently have a thickened basement membrane / peritubular hyalinization and lack spermatogenic maturation
* Can spread in pagetoid fashion into rete testis, along the plane between the rete epithelium and the basement membrane

Molecular:
* Aneuploid (polyploid) with gain of additional findings at the time of invasion (e.g., isochromosome 12p is seen in invasive disease but not in GCNIS)
* loss of FIT expression –> FHIT gene, located at 3p14.2

IHC:
OCT 3/4
SALL4
Podoplanin / D2-40
KIT / c-KIT / CD117
PLAP

Question 7

Seminoma

Answer 7

• Most frequent germ cell tumor of the testis
• Presents in young men (30 - 49) with unilateral palpable mass
• Typically a well demarcated, uniform neoplasm with characteristic cytological features and background of fibrous septae and lymphocytes
• Serum LDH and PLAP may be elevated
• hCG levels are increased in up to 20% of cases but elevation is modest
• AFP should not be increased, if elevated think liver disease or nonseminoma component

Macro:
* Well demarcated, homogeneous, solid cream or grey tumors; surface nodularity and lobulation; may be multiple nodules
* Necrosis or hemorrhage usually minimal
* If regressed, only a scar may be visible
* Usually confined to testis (90%)

Micro:
* Sheets or lobular configuration of tumor with fibrous septae
* Cells are typically pale (glycogen) but may be eosinophilic
* Cell membranes are well defined with distinct cell boundaries
* Nuclei are polygonal and may have a flat edge giving a squared off appearance; they contain one or more prominent central nucleoli; no nuclear overlap if well fixed tissue
* A lymphocytic infiltrate is present (T lymphocytes) with plasma cells; germinal centers may occur
* Granulomas noted in up to 50% of cases
* Intercellular edema with microcystic spaces and coagulative type necrosis can be present
* Multinucleated syncytiotrophoblasts can be seen in 20% of tumors and can produce hCG

Molecular:
* Isochromosome from the short arm of chromosome 12, i(12p) present
* KIT mutations

IHC:
OCT 3/4 (100% nuclear)
CD117 –> KIT (90-100% cytoplasmic membrane)
D2-40 –> podoplanin (100% cytoplasmic)
PLAP (86-95% cytoplasmic membrane)
SALL4 (100% nuclear)
PAS

CD30 and AFP negative –> differentiated between embryonal carcinoma and yolk sac tumour

Question 8

Embryonal carcinoma

Answer 8

• Pleomorphic, high grade appearing type of GCT
• Usually occurs mixed in combination with other types of testicular GCT
• Predominance in a mixed GCT is associated with a higher risk of metastases and relapse
• Elevated serum human chorionic gonadotropin (hCG), alpha fetoprotein (AFP) and lactate dehydrogenase (LDH) may be present
• Serum tumor markers can be used to monitor for recurrence

Macro:
* Gray-tan mass with hemorrhage and necrosis

Micro:
* Multiple growth patterns usually present
* 3 most common growth patterns: solid (55%), glandular (17%) and papillary (11%)
* Polygonal cells
* Cells are crowded, have indistinct distinct cell borders and appear to have overlapping nuclei
* Pleomorphic, high grade nuclear features
* Mitotic figures are common
* Necrosis is common, both as single cell necrosis and larger foci
* Often grows admixed with yolk sac tumor and can rarely form polyembryoma-like structures, called embryoid bodies
* Residual seminiferous tubules may contain germ cell neoplasia in situ (GCNIS) or intratubular embryonal carcinoma, which entirely fills the tubule
* Lymphovascular invasion is common within embryonal carcinoma predominant GCTs, is usually best visualized at the periphery testicle and may entirely occlude vessels, mimicking nodules of tumor

Molecular:
* Isochromosome 12p: present in most testicular GCT components including embryonal carcinoma

IHC:
* Positive
OCT 3/4 (nuclear and cytoplasmic)
CD30
PLAP: heterogenous/patchy and weak
SOX2
AE1 / AE3
SALL4

• Negative
  AFP
  HCG
  CD177
  EMA

Question 9

Yolk sac tumor

Answer 9

• Germ cell neoplasm composed of cells / structures reminiscent of embryonic / fetal yolk sac, allantois and extraembryonal mesenchyme
• Elevated serum alpha fetoprotein (AFP) should raise the suspicion for a yolk sac component

2 biologically distinct entities:
* Postpubertal type (germ cell neoplasia in situ [GCNIS] related and usually as part of mixed tumor)
* Prepubertal type (unrelated to germ cell neoplasia in situ and usually pure)

Macro:
Postpubertal type:
* Poorly circumscribed, nonencapsulated, predominantly solid
* Gray to white to yellow to tan, gelatinous surface
* Hemorrhage, necrosis and cystic areas are common

Prepubertal type:
* More homogeneous, gray to yellow, solid or mucoid, bulging and lobulated
* Hemorrhage and necrosis are uncommon

Micro:
Microcystic / reticular pattern:
* Most common
* Anastomosing cords of flattened cells forming a honeycomb / spider web meshwork, forming spaces enclosing mucoid / basophilic material

Other patterns:
* Solid
* Myxomatous
* Endodermal sinus
* Papillary
* Glandular
* Macrovesicular
* Polyvesicular vitelline
* Hepatoid
* Sarcomatoid

–

• Cytological atypia is variable across patterns; it is most significant in solid, sarcomatoid and glandular patterns
• 50% of tumors contain Schiller-Duval bodies (papillary structures within cystic spaces, lined by cuboidal to columnar cells with a distinct central vessel)
• Characteristic (but not pathognomonic) hyaline globules (within and outside cytoplasm), refractile and eosinophilic, which are alpha-1 antitrypsin+ and PAS diastase+

Molecular:
Postpubertal type:
* Gain of 12p (usually isochromosome 12p)
* Erased genomic imprinting

Prepubertal type:
* Multiple gains (1q, 20q, 22) and losses (1p, 4, 6q) but no 12p gain
* Biparental genomic imprinting

IHC:
AFP (more specific but less sensitive: variable, often focal, in many cases entirely absent)
Glypican 3 (more sensitive, staining almost all cases but less specific)
SALL4 (sensitive and specific for most germ cell tumor types including yolk sac tumor)
Pancytokeratin (uniformly positive)
Villin (usually positive)
CK7 (84%)

Negative:
PLAP, OCT3/4, CD30, CD117, PLAP, D-40

Question 10

Teratoma

Answer 10

• Tumor originating from germ cells with more than one embryonic germ layer
• Prepubertal and postpubertal teratoma: currently accepted categories of teratoma
• Either category may occur in either age group

Macro:
* Lobulated, with cysts of mucinous, gelatinous or serous material

Micro:

Postpubertal type
* Demonstrate varying degrees of atypia
* Any type of tissue may be present, such as gastrointestinal glands, respiratory epithelium, cartilage, squamous epithelium with keratinization, primitive undifferentiated spindle cells, or neuroepithelium
* Will have GCNIS
* Associated with atrophic testis with sclerosis and microliths
* and muscularis propria) but any tissue type may be present
* Hair follicles not seen

Prepubertal type
* More likely to have tissue arrangements mimicking organs (organoid morphology with tissue layers visible such as epithelium, lamina propria and muscularis propria) but any tissue type may be present
* Hair follicles may be seen (not in postpubertal type)
* No cytologic atypia, GCNIS or necrosis; minimal mitoses

Teratoma with somatic type malignancy
* Virtually only in post-pubertal patients
* Expansile or infiltrative overgrowth of teratomatous elements
* Sarcoma is most prevalent somatic type malignancy but other tumor types occur, including adenocarcinoma, squamous cell carcinoma and primitive neuroectodermal tumor (PNET)

Molecular:
Postpubertal type
* Associated with germ cell neoplasia in situ (GCNIS)
* Chromosome 12p amplification
* Thought to arise from GCNIS

Prepubertal Type
* Associated with GCNIS
* Not associted Chromosome 12p amplification
* Prepubertal type significantly less likely to metastasize or recur

Question 11

Spermatocytic tumor

Answer 11

• Spermatocytic tumor (ST) is a polymorphous triphasic germ cell
• Does not arise from germ cell neoplasia in situ
• More common in older patients compared with patients with other germ cell tumors
• Classically characterized by tripartite tumor cells with 3 different cellular sizes (small, intermediate, giant)
• Serum markers not elevated
• Indolent clinical behaviour
• Cryptorchidism is not a risk factor
• 9% bilateral (synchronous or more frequently, metachronous)

Macro:
* Homogenous or lobulated nodule
* Grayish white
* Edematous or mucoid
* Soft
* Necrosis and hemorrhage can be present
* Extension beyond the tunica albuginea might be present

Micro:
* Landmark feature - tripartite cytology with cells of 3 different sizes:

Small cells (6 - 8 microns)
* Round dark nuclei
* Narrow rim of eosinophilic cytoplasm
* They resemble lymphocytes

Intermediate size cells (15 - 20 microns)
* Round nuclei with granular to filamentous chromatin (so called spireme-like chromatin, after the thread-like chromatin at the beginning of prophase)
* Usually no evident nucleoli, although they might be present
* Dense eosinophilic to amphophilic cytoplasm with no glycogen
* Poorly defined cytoplasmatic membranes
* Clearer cells with distinct membranes can be found close to edematous areas

Giant cells (50 - 150 microns)
* 1 or more nuclei with the same features of medium cells
* Eosinophilic cytoplasm with the same features of medium cells
* Size distribution can vary among different cases (usually medium cells > small cells > giant cells)
* Mitoses (even atypical) and apoptotic bodies can be abundant
* Stroma is scant, with only small strips of connective tissue

Molecular:
* Classically without 12p abnormalities
* Presence of 12p amplification (hybrid genetics) might be linked to the development of metastases or anaplastic morphology

IHC:
Positive:
CD117
SALL4
DMRT1
NUT (specificity 100%, sensitivity 41% when positivity is present in ≥ 50% of neoplastic cells with equal intensity to spermatogonia internal control)
OCT2
SSX2

Negative:
EMA, PLAP, OCT3/4, beta subunit of hCG, AFP, CEA, CD30
Differentiates it from seminoma/lymphoma

Question 12

Germ Cell IHC Summary

Answer 12

Question 13

Germ Cell Serum Marker Summary

Answer 13

Question 14

Leydig cell tumor

Answer 14

• Most common sex cord stromal tumor of the testis; comprised of cells resembling nonneoplastic Leydig cells
• A small minority (< 10%) of cases are clinically malignant
• Mostly sporadic, rarely associated with hereditary leiomyomatous and renal cell carcinoma syndrome
• Occurs at any age with 2 peaks: 5 - 10 years and 30 - 60 years
• Usually unilateral, rarely bilateral
• Painless testicular enlargement

Children
* Precocious puberty caused by androgen production
* Gynecomastia and breast tenderness due to estrogen production

Adults
* Gynecomastia
* Infertility, loss of libido and erectile dysfunction
* Rarely, Cushing syndrome

Macro:
* Well circumscribed, solid homogeneous mass
* Golden brown or brownish green cut surface
* Hyalinization and calcification may be present

Gross features suggestive of malignancy:
* Large size: > 5 cm
* Infiltrative margins
* Hemorrhage and necrosis
* Extratesticular extension

Micro:
Architecture:
* Typically arranged in sheets
* Uncommon patterns: insular, trabecular, pseudotubular, ribbon-like, trabecular, spindled and microcystic

Cytologic features:
* Polygonal cells with abundant eosinophilic granular cytoplasm, uniform round nuclei and prominent central nucleoli; rarely, nuclei may have a ground glass appearance
* Uncommon cell types: scant cytoplasm, foamy cytoplasm and spindling
* Lipofuscin pigment maybe present: golden yellow on H&E stain, red-purple granular appearance on PAS stain
* Binucleated and multinucleated cells may be present
* Reinke crystals: pathognomonic; identified in only up to 30% (degradation / dissolution by formalin fixation); intracytoplasmic, rarely extracellular
* Mitosis: rare

Molecular:
* Gain of chromosome X, 19 or 19p and loss on chromosome 8 and 16 are most frequent findings
* Somatic GNAS (guanine nucleotide binding protein, alpha stimulating activity polypeptide 1)

IHC:
Positive:

Inhibin A
calretinin
MelanA
androgen receptor (AR)
steroidogenic factor 1 (SF1)
Insulin-like 3 (INSL3) variably reported to be positive
CD99 (MIC2)

Negative:

PLAP
SALL4
EMA

Question 15

Sertoli cell tumor

Answer 15

• Sex cord stromal tumor of testis composed of cells showing features of fetal, prepubertal, adult or atrophic Sertoli cells, at least focally
• Second most common type of pure sex cord stromal tumor after Leydig cell tumor
• Most are small, unilateral tumors in adults and the vast majority are benign
• Risk factors: cryptorchidism, familial adenomatous polyposis
• Vast majority are benign, ~ 5% cases are malignant
• Tumors with sclerosis > 50% almost invariably benign
• Large cell calcifying variant —> <20 yo, component of Carney syndrome
• Intratubular large cell- hyalinizing sertoli cell neoplasia —> associated with Peutz-Jeghers syndrome

Features associated with malignancy:
* extratesticular spread, size > 5 cm
* lymphovascular invasion
* high grade cytological atypia
* tumor necrosis
* mitotic index > 5/10 high power fields

Macro:
* Cut surface usually homogenous white or yellowish
* Cystic component present in ~ 30% of cases
Benign: well circumscribed, most between 2 - 5 cm (Am J Surg Pathol 2014;38:510)
Malignant: > 5 cm, poor circumscription, extratesticular extension, necrosis, hemorrhage

Micro:
* Architecture: tubular (lumen), cords (no lumen), tubulopapillary present at least focally
* Other patterns: macro or microcystic, nested, trabecular, whorled, solid, retiform, pseudopapillary; most tumors show mixed patterns
* Nuclei: bland, uniform, round to ovoid in benign tumors; small hyperchromatic or large with prominent nucleoli in malignant tumors
* Cytoplasm: usually clear and abundant but extremely variable, ranging from scant, foamy, eosinophilic to markedly lipidized; hyaline globules are common
* Stroma: variable, basement membrane-like material around tubules, sclerotic, myxoid, edematous, angiomatous
* If sclerosis > 50% → sclerotic variant (now considered subtype of Sertoli cell tumor, NOS)

Molecular:
* CTNNB1 gene mutations
* Negative for isochromosome 12p

IHC:

Positive stains
S100
Inhibin
Cytokeratin
Vimentin
Calretinin

Negative stains
SALL4, OCT4, PLAP, alpha fetoprotein, hCG, CD30

Question 16

DLBCL-primary testicular lymphoma

Answer 16

• Primary testicular lymphoma is rare
• Lymphoma –> 5% all testicular tumours
• Typically presents in adult men with a median age between 60 and 70 at diagnosis
• Most common testicular malignancy in men > 60 years of age
• Most common testicular malignancy to present with bilateral involvement
• Increased incidence in HIV positive men
• HIV positive patients with primary testicular diffuse large B cell lymphoma (DLBCL PT) are younger (median age of 36)

Macro:
* Enlarged testicle with a fleshy, diffuse or lobulated firm mass
* Cut surface is relatively homogeneous and bulging, with a tan-yellow color
* Often demonstrates a sharp demarcation from testicular parenchyma

Micro:
* Diffuse sheets of large lymphoma cells
* Initially infiltrate between seminiferous tubules before total replacement of the parenchyma
* Lymphoma cells frequently invade and fill the seminiferous tubules
* Frequent mitotic figures can be seen
* Necrosis is common
* Lymphoma cells show centroblastic morphology in most cases (90%) and less commonly, immunoblastic morphology (10%)
* Associated changes in the testis include spermatogenic arrest, interstitial fibrosis and tubular hyalinization

Molecular:
* IGH rearrangements in virtually all cases
* BCL2 and MYC rearrangements occur in 10% and 15% of cases, respectively
* BCL6 rearrangement occurs in 40%

IHC:
Pan B cell antigens (CD19, CD20, CD79a, PAX5)
BCL6 (75 - 90%), MUM1 (40 - 70%), CD10 (20 - 40%)
TCL1 (~67%), FOXP1 (~80%)
BCL2 (~75%), MYC (13%)

Question 17

Gonadoblastoma

Answer 17

• Rare benign gonadal tumor composed of germ cells and sex cord stromal derivatives resembling immature granulosa cells and Sertoli cells
• Most cases occur within first 2 decades; nearly 94% by age 30
• Almost always associated with disorders of sex development
• Occurs in –> Frasier syndrome, Swyer syndrome, Turners syndrome
• Excellent prognosis prior to malignant transformation, with no deaths reported
• 30% of patients have malignant germ cell tumor in dysgenetic or contralateral gonad

Macro:
* Tumor size varies from microscopic to 8 cm
* Yellow to tan nodules with gritty cut surface
* No hemorrhage or necrosis unless transformation to malignant germ cell tumor

Micro:

Three growth patterns
* Most common: round / irregular clusters of immature Sertoli cells and germ cells surrounded by basement membranes, with Sertoli cells encircling rounded hyaline nodules
* Sertoli cells surround large germ cells
* Germ cells occupy center of nests with peripheral ring of Sertoli cells
–

• Stroma may contain large polygonal cells indistinguishable from
• Leydig cells: common in postpubertal patients
• Calcification common: may be focal involving hyaline bodies or extensive
• 50% have coexisting or subsequent seminoma
• 8 - 10% develop other germ cell malignancies

Molecular:
* TSPY gene is most likely candidate

IHC:
* Germ cells express p53 , PLAP, CD117 / c-kit,
* Stromal cells express inhibin, WT1