how does immunohistochemistry plays role in testitucular tumors, its hostoly as well.

1. ROLE OF IHC IN
TESTICULAR TUMORS
BY: DR. PRERNA CHOUDHARY

2. TESTIS- ANATOMYandHISTOLOGY
Measures- 4.5 X 2.5 X 3 cm ; weighs:20 gram

3. INTRODUCTION
•Testicular cancers are the most common solid malignancy and the second leading
cause of cancer death in young men
•Affecting males between the ages of 15 and 35, although they represent only
approximately 1 percent of all solid tumors in men
•The are classified as per WHO CLASSIFICATION OF TUMORS OF TESTIS 2016
•THE H&E is gold standard for diagnosis, IHC plays a significant role and <5% of tumors
require Immunostaining for accurate interpretation.
•Differential diagnosis can be resolved with IHC
•BUT, few pre-analytical errors to be kept in mind

5. PREANALYTICAL ERRORS
IHC is very important for diagnosis of type of cancer, however there can few errors which give
false results
FALSE NEGATIVE:
Antibody inappropriate, denatured , used at wrong concentration
Loss of antigen through poor fixation/diffusion.
Presence of antigen at a density below level of detection.
FALSE POSITIVES:
Cross- reactivity of antibody with unintended antigens
Nonspecific binding of the antibody to the tissue
Presence of endogenous peroxidase
Entrapment of normal tissues by tumor cells

6. GERM CELL TUMORS

9. Non-invasive GCN

10. GERM CELL NEOPLASIA IN SITU
•Syn – Intratubular germ cell neoplasia, unclassified;
testicular intraepithelial neoplasia
•Derived from primordial germ cells/gonocytes that failed to
differentiate into spermatogonia
• Adult testis – inside seminiferous tubules, between sertoli
cell and basal membrane;
•infantile testis - GCINS cells detached from basal membrane
•No. tubules affected – few to 100% (variable)
•Gonocyte-like germ cells - clear cytoplasm; angulated nuclei;
coarse chromatin ,base of tubules
•Nuclei - string of beads – separated from the lumen of
tubule – by uniform sertoli cell nuclei

11. IHC – PLAP (cytoplasmic), OCT3/4(NUCLEAR), Podoplanin, NANOG, LIN28 (absent – normal
spermatogonia), SALL4
KIT(CD117) – non specific; normal spermatogonia – may be positive (cytoplasmic
membrane)
Frozen section – Alkaline phosphatase – helpful
EMA, CD30, GLYPICAN 3, SOX2

12. •DD
Vacolization of seminiferous tubule cells (degenerative change)
Maturation arrest in spermatogonia
Metastatic carcinoma- melanoma, lymphoma may colonize seminiferous tubules
OCT ¾ - resolve this matter

13. Marker with normal tissues expressing type dd
Oct ¾ (Octamer-binding transcription
factor 4)
Is not expressed in normal,
differentiated cells
nuclear • Highly sensitive and specific for seminoma
• Poor prognostic factor for esophageal squamous cell
carcinoma
• May be poor prognostic factor for non-small cell lung
cancer
• MAY LOOSE REACTIVITY AFTER CHEMOTHERAPY
PLAP (Placental Alkaline Phosphatase)
placenta
Cytoplasmic • Germ cell tumors(does not stain spermatocytic seminoma)
• Intratubular germ cell neoplasia
• Trophoblastic tumors
• Serum PLAP is increased 10 fold in smokers- so not always
included in diagnostic workup
Podoplanin (D2-40)
Novel marker of mesothelial cells,
germ cells, lymphatic endothelial cells
membranous Seminoma (100%)
Embroyonal carcinoma (30%)
Others: mesothelioma
dermatofibroma
Hemangioblastoma
Primary skin adnexal tumor

14. Marker with normal tissues
expressing
type dd
SALL4
Embryonic stem cells
Nuclear Pan germ cell tumor marker
CD117(stem cell factor receptor)
Germ cells
Cytoplasmic and membranous • Seminoma
• Intratubular germ cell neoplasia
Others: Gist
PEComas
Oncocytomas
CD 30 Membranous and golgi(paranuclear
dot like)
Embryonal carcinoma

15. Specificforms – Intratubulargermcellneoplasia
Intratubular seminoma – complete filling of tubules
by neoplastic cells (identical to seminoma, GCINS),
lymphocytes present
IHC – simillar to seminoma
Intratubular non – seminoma – almost always
embryonal carcinoma
Distortion of tubules , enlarged beyond normal
diameter, necrosis, calcification
IHC – OCT3/4 +, CD 30 +, CD 117 –
(CD 30 is negative in GCNIS)

16. INVASIVE GCN- single histological type

17. Seminoma
•Malignant germ cell tumour - considered - neoplastic
counterparts of the primordial germ cells /gonocytes present
during early embryonic development
•Syn – geminoma; ovary - dysgerminoma
GROSS MICRO:

19. IHC –
Positive – OCT3/4, SALL4, CD117, D2-40 (podoplanin),
SOX17, PLAP
Negative - HMW Keratin, CD30, SOX2, GATA3,
Glypican-3,
PLAP

20. Differential diagnosis
1. Solid pattern embryonal ca –
seminoma – prominent lymphocytic infiltrate;
+ve for KIT, Podoplanin, SOX17 ;
-ve for CD30, SOX2, Cytokeratin AE1/AE3
2. Solid yolk sac tumour -
yolk sac tumour patterns, less lymphocytes, and absence of fibrous septa
-ve for OCT3/4;
+ve for AFP ,glypican 3 & cytokeratin AE1/AE3
3. Choriocarcinoma
seminoma with syncytiotrophoblast – syncytiotrophoblast associated with seminoma
cells and not mononucleated trophoblasts (chorioca)
4. spermatocytic tumor
5. Malignant Sertoli cell tumor

21. Embryonalcarcinoma
•Malignant germ cell tumour composed of tumour
cells resembling embryonic stem cells
•Gross – Histo
•Patterns - Solid, glandular and papillary etc
combinations of two pattern - frequent

22. Positive
OCT3/4, SALL4, CD30, AE1/3, SOX2
Negative
CD117, D2-40, Glypican-3 , vimentin, podoplanin

23. Marker with normal tissues expressing type dd
Sox 2
Fetal CNS tissue
nuclear Embryonal carcinoma
PNET in teratoma
AE1/AE3 (pan cytokeratin cocktail)
Most epithelial cells
cytoplasmic Used with conjugation with CAM5.2
to screen carcinomas

24. Yolksac tumour, postpubertal-type
•Malignant GCT that differentiates to resemble
extraembryonic structures- yolk sac, allantois,
and extraembryonic mesenchyme
•Gross

25. •Microscopy – various patterns –microcystic, reticular, macrocystic, endodermal sinus pattern
(Schiller- Duval bodies) papillary, glandular, myxomatous
•Uncommon – polyvesicular vitelline, hepatoid and Intestinal
•Intracytoplasmic and extracellular hyaline globules (refractile eosinophilic)
•Band-like intercellular deposits of basement membrane - parietal differentiation.

28. IHC –
Positive - Glypican-3, SALL4, AE1/3, GATA-3, AFP(V),
PLAP (V)
Negative OCT4, CD30,hCG

29. Marker with normal tissues expressing type dd
Glypican – 3
By- embroyonic liver, placenta
(syntiotrophoblast)
Cytoplasmic,
membranous,
canlicular
HCC
Hepatoblastoma
Yolk sac tumor
Choriocarcinoma
Placental ste trophoblastic tumor
AFP
Fetal liver
Cytoplasmic Yolk sac tumor
OTHERS :HCC
HEPATOBLASTOMA
GATA -3
Involved in the luminal differentiation of
breast epithelium, development of
collecting system / urothelium and
trophoblastic differentiation
nuclear renal tumors
Paragangliomas
Choriocarcinomas
Mesotheliomas
yst

30. TROPHOBLASTICTUMOR
Choriocarcinoma
Malignant GCT that differentiates to resemble the
trophoblastic cells of the extraembryonic chorion –
cytotrophoblast, intermediate trophoblast, and
syncytiotrophoblast
Gross - Micro -

31. Positive - b- HCG, SALL4, Glypican-3,
AE1/3, GATA-3,PLAP
Negative - OCT4, CD30

32. Non-choriocarcinoma trophoblastic tumors:
•GCTS showing trophoblastic differentiation other than choriocarcinoma and
non-trophoblastic contaning syntiotrophoblasts
• Cystic trophoblastic tumor – post therapy
• Epithelioid trophoblastic tumour
• Placental site trophoblastic tumour,
• Regressing choriocarcinoma,
• Hybrid trophoblastic tumours.
Positive stain - Beta hydroxysteroid dehydrogenase, Inhibin, GATA 3 & CK 18

33. Marker with normal tissues expressing type dd
Hcg (human chrionic gonadotrophin)
syntiotrophoblast
cytoplasmic • Choriocarcinoma
• Syntiotrophoblast gaint cells in
seminoma
Inhibin
Granulosa cell, Sertoli cells, adrenal cortical
cells, trophoblasts
cytoplasmic • Adrenocortical neoplasm
• Sex cord stromal tumors
• Trophoblastic tumors
• Hemangioblastoma

34. Teratoma
postpubertal-type
Malignant GCT composed of several types of tissue
representing one or more of the germinal layers
endoderm, mesoderm & ectoderm
syn – mature teratoma, immature teratoma
Gross
Micro - any epithelial or mesenchymal tissue type can
be seen, as can neural tissue.; cytological atypia

35. • IHC- glandular element- epithelial membrane antigen, HCG (syntiotrophoblast), PLAP
(glandular structures)
Histologically different from prepubertal
Solid appearance
Disordered arrangement of tissues
Atypia and mitosis
Associated to GCNIS (Primary criteria for distinction between pre and postpubertal)

36. TERATOMAS WITH SOMATIC TYPE MALIGNANCY
•Develops a distinct secondary component that resembles somatic-type malignant
neoplasm (eg. Sarcomas and carcinomas)
•GROSS- grey white nodule, with necrosis and haemorrhage
•HISTO : Expansile/infiltrative growth patterns. Organoid admixtures of varied elements
admixed with GCT components.
• PNET another m/c to be found
• Adult type: Neuroendocrine tumors (carcinoids), carcinomas.
• IHC – similar to the counter parts in other organs
PLAP, OCT3/4, ALPHA FETO protein

37. TABLE

38. Mixedgermcell tumour
•Contains 2 or more malignant GCT components
•Most frequent combination: Embryonal Ca with teratoma,
seminoma and/or yolk sac
•Combination of prepubertal teratoma and yolk sac tumor is
not considered mixed GCT
•Gross – variegated appearance depending upon the
components present

39. MICRO:
A cystic glands of embryonal carcinoma with a more subtle
microcystic yolk sac tumour component.
B Polyembryoma variant of a mixed germ cell tumour forms
embryo-like bodies with a core of embryonal carcinoma, an
amniotic-like space, and a "ventral" yolk sac tumour component
C Diffuse embryoma. A diffuse embryoma variant of a mixed
germ cell tumour shows a necklace-like arrangement of
columnar embryonal carcinoma cells with a parallel component
of flat yolk sac tumour cells.

40. IHC – {{ OCT3/4 (Embroynal ccomponent
positive,yolk sac negative) , CD30, CK 7, NANOG}} ,
GLYPICAN 3, cd117(yst), SALL4(both +),
B-HCG

41. PUT ALGORITH

42. GERM CELL TUMORS UNRELATED
TO GERM CELL NEOPLASIA IN SITU

43. Prepubertal type GCN

44. SPERMATOCYTIC TUMOR
•From post pubertal type germ cells
•FGFR3 and HRAS gene mutation
•IHC-SALL4, CD117(50%), NUT(nuclear protein in testis), GAGE7, NY-ESO1, GAGE1, MAGEA 3, MAGEA 4, MAGEC1
• OCT4, CD30, AFP, PLAP, hCG, calret

45. TABLE OF DIFFRENCE BETWEEN 2

46. TERATOMA, PREPUBERTAL- TYPE
Composed of somatic tissues derived from one or more germinal layer.
SIMILAR TO MATURE CYSTIC TERATOMA OF OVARY
Gross and micro:

47. MIXED TERATOMA AND YOLK SAC TUMOR,
PREPUBERTAL
Gross- resembles prepubertal teratoma
Micro-
IHC- AFP (YST COMPONET),

48. Pre pubertal Yolk Sac tumor
m/c tesisticular GCT in infants and children
Histologically, identical to postpubertal type
SAME IHC PROFILE AS POST-PUBERTAL YST

49. SEX CORD-STROMAL TUMORS
2-5% Of testicular neoplasm

50. LEYDIG CELL TUMOR
Gross-
REINKE CRYSTAL,
LIPOFUSCHIN
PIGMENT

51. IHC- lnhibin, calretinin, SF1 , melan A, and C D99 are
typically positive. Chromogranin , synaptophysin, and cytokeratins are variably expressed, and
< 10% of tumours are S 1 00 protein-positive
Dd- Congenital adrenal hyperplasia (testicular adrenal rest tumours)

52. SERTOLI CELL, NOS
is a neoplasm composed of sex cord cells that usually shows focal tubular differentiation
but rarely grows diffusely.
GROSS- 2-5cms, well circumscribed with uniform yellow cut surface
HISTO-
IHC- Inhibin (50%) nuclear beta catenin (60-70%) , calretinin, SF1, CD99, melan A, WT1
vimentin, s100, sox9 (few)

54. LARGE CELL CALCIFYING SERTOLI CELL TUMOR
Composed of abundant eosinophilic cytoplasm and focal to massive calcifications.
PRKAR1A germ line mutation (60-70%)
GROSS- 1-15cms, tan to white, gritty, necrosis and haemorrhage
HISTO-
IHC- INHIBIN, s100, SF1, beta-catenin

55. INTRATULAR LARGE CELL HYALINIZING SERTOLI CELL NEOPLASIA
Occurs exclusively in paitents with Peutz – Jeghers syndrome
GROSS- Scattered firm foci measuring few mm in diameters
HISTO-
IHC- Inhibin, aromatase, cytokeratin

56. GRANULOSA CELL TUMOR- ADULT
•Neoplasm composed of sex cord cells that resemble granulosa cells of Graafian follicles of
ovary.
GROSS- well circumscribed, solid,sometimes cyst, c/s- lobulated yellowish tan.
MICRO-
IHC- VIMENTIN, calretinin , CD99, melan A, FOXL2, smooth muscle actin, S100
nuclear beta catenin

57. JUVENILE GRANULOSA CELL TUMOR
Shows solid and follicular patterns with immature nuclei
GROSS- well circumscribed, solid, cystic , yellow to tan-white
MICRO-
IHC- Inhibin, calretinin, CD99, SOX9, VIMENTIN, PANCYTOKERATIN, SMOOTH MUSCLE ACTIN

60. TUMOR CONTAINING BOTH GERM CELL AND
SEX CORD STROMAL TUMOR ELEMENTS
GONADOBLASTOMA
Arise from dysgenetic gonad
Restis-specific protein Y-encoded (TSPY) gene involved
GROSS-granular, gritty c/s- calcifications
HISTO- Spindle cell gonadal stroma , occasional
Leydig like cells lacking reinke crystal.

61. IHC-OCT3/4, FOXL2 (does not stain Sertoli cells of GCNIS THUS DIFFRENTIATE FROM
GCNIS) SEC CORD CELLS ( staing for alpha-inhibin, calretinin, FOXL2) and absence of
infiltrating lymphocytes distingyush from dysgerminoma/seminoma.

62. HEMATOLYMPHOID TUMORS

65. METASTATIC TUMOR
Secondary tumours coming from direct spread or distant metastasis

66. REFERENCES: