2013 International Society of Urologic Pathology
Conference on Best Practices Recommendations in
the Application of Immunohistochemistry in
Diagnostic Urologic Pathology: The Role of
Immunohistochemistry in Testicular Neoplasms
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Thomas M. Ulbright, MD
Daniel M. Berney, FRCP
Satish K. Tickoo, MD
John R. Srigley, MD
Principles of IHC in Neoplasms of the Testis
• There are recurring differential diagnostic
situations in testicular neoplasia
• IHC is not a diagnostic tool but a differential
diagnostic tool
• Clinical, serological and light microscopic features
are critical to the generation of reasonable and
limited numbers of differential diagnoses
• Exceptions to expected IHC reactivities are
invariable and emphasize the need to correlate IHC
with morphologic and clinical features
• If a pathologist cannot narrow the differential
diagnostic considerations to 2 or 3 entities without
IHC, “expert” consultation should be sought
Principles of IHC in Neoplasms of the Testis (con’t)
• IHC is very helpful to resolve between
differential diagnostic considerations and
should be applied in a conservative fashion,
ideally utilizing 2 or 3 immunostains with
different patterns of reactivity for the
differential diagnoses under consideration
• Diagnostic algorithms for specific differential
diagnostic considerations should therefore
be utilized
• There are currently no clinically utilized
prognostic IHC markers for testicular
neoplasms.
Useful Antibodies for Testicular Neoplasms:
OCT4 (OCT3/4, POU5F1)
• Nuclear protein critical for pluripotency of
embryonic stem cells
• ~100% sensitive for IGCNU, seminoma,
embryonal carcinoma (EC)
• Negative in other testis tumors
• Other + tumors: rarely, lung & kidney ca, large
cell lymphoma
• Caveat: Post-chemo ECs may be negative
• Overview: Very valuable for seminoma vs
mimics (yolk sac tumor [YST], Sertoli cell
tumor [SCT]) and to support Dx of seminoma
and EC in Bxs (mets).
Useful Antibodies for Testicular Neoplasms:
CD117 (cKIT)
• Receptor tyrosine kinase in stem cells
• Membranous expression occurs in 95-100% of
seminomas & IGCNU
• Variably + in YSTs and spermatocytic
seminomas (SS)
• ~ negative in ECs & choriocarcinomas (CC)
• Other + tumors: Numerous
• Caveat: Spermatogonia may be +; therefore not
helpful for IGCNU
• Overview: Main utility is assisting with the Dx
of seminoma vs EC
Useful Antibodies for Testicular Neoplasms:
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Podoplanin (D2-40, M2A)
Transmembrane mucoprotein expressed on fetal
germ cells, lymphatic endothelium & mesothelium
~100% of IGCNU & seminomas show membranous
positivity
YSTs, CCs, SSs, non-neoplastic GCs ~negative
Other + tumors: Numerous (gliomas,
meningiomas, mesothelial, lymphatic, adenocas
etc.)
Caveat: Up to 30% of ECs show some positivity
limited to apical surfaces
Overview: Useful for seminoma vs solid YST
(unlike CD117) and EC (beware of apical staining).
Better for IGCNU than CD117
Useful Antibodies for Testicular Neoplasms:
SOX17 (SRY-box 17)
• Member of the SOX family of nuclear
transcription factors involved in embryonic
development
• Positive in seminomas (95%), IGCNU (~100%),
YSTs (50%), some teratomas
• Negative in ECs, CCs & SSs (with limited
experience in CC & SS).
• Other + tumors: ?
• Caveat: Non-neoplastic germ cells are +
• Overview: Mostly useful for seminoma vs EC.
Not helpful for IGCNU. May become best marker
for seminoma vs EC but more experience
needed
Useful Antibodies for Testicular Neoplasms:
CD30 (Ki-1,Ber-H2)
• Protein in the TNF receptor family with
membranous and Golgi-zone staining
• 93-100% of ECs are positive
• Negative or, at most, stains rare cells in other
GCTs
• Other + tumors: lymphomas, soft tissue tumors,
melanomas & infrequent carcinomas
• Caveats: Intensity in EC can be variable
requiring close examination. Loss occurs in
some post-Rx ECs.
• Overview: Very useful for EC vs seminoma or
solid YST.
Useful Antibodies for Testicular Neoplasms:
AE1/AE3 cytokeratin
• Antikeratin cocktail specific for CKs 1-8, 10, 14, 15,
16 & 19.
• Generally positive (cytoplasmic) in all nonseminomatous tumors
• Usual seminomas are mostly non-reactive, with
some positivity in 20-36%, mostly paranuclear &
dot-like.
• Other + tumors: Numerous (carcinomas & others)
• Caveat: Marks trophoblast cells in seminomas, &
these cases are still seminomas.
• Overview: Widely available Ab mostly helpful to
distinguish seminoma vs. EC
Useful Antibodies for Testicular Neoplasms:
SOX2 (SRY-box 2)
• Member of the SOX family of nuclear
transcription factors involved in embryonic
development; needed for pluripotency of
undifferentiated embryonic stem cells
• Positive in 96% of ECs & <1% of seminomas
• Negative in YSTs, CCs & IGCNU
• Other nuclear + tumors: immature elements in
teratoma, melanoma & rhabdoid tumors
• Caveat: Non-neoplastic Sertoli cells are +
• Overview: Mostly useful for seminoma vs EC.
May become a preferred marker for seminoma vs
EC but the panelists considered it currently
technically difficult
Useful Antibodies for Testicular Neoplasms:
Glypican 3 (GPC3)
• Membrane anchored heparan sulfate
proteoglycan
• Positive in YSTs (100%), CCs (80%), teratomas
(“immature”) (17%) & rare ECs (5%)
• Negative in IGCNU & seminoma
• Other + tumors: : hepatocellular and gastric
cancers
• Caveats: Syncytiotrophoblast cells are often
positive (71%)
• Overview: More sensitive but less specific for
YST among testis GCTs than AFP
Useful Antibodies for Testicular Neoplasms:
Alpha-fetoprotein (AFP)
• A major plasma protein produced by the yolk sac &
liver during fetal life
• YSTs are variably and often focally positive (overall,
~80%)
• AFP is negative in the other GCTs, except for glands
and luminal secretions of some teratomas
• Other + tumors: hepatocellular neoplasms, hepatoid
carcinomas & occasional other non-GCTs
• Caveat: Negative AFP does not exclude YST
• Overview: Wide availability & relative YST specificity
make it helpful for Dx of YST but has limited
sensitivity
Useful Antibodies for Testicular Neoplasms:
Human Chorionic Gonadotropin (hCG)
• A dimeric glycoprotein produced by placental
trophoblast cells, mostly syncytiotrophoblasts; α
subunit is shared by LH, TSH & FSH but the β is
unique
• Primary CC is positive (100%) for βhCG as are all
non-CCs with syncytiotrophoblast cells
• Other + tumors: any non-germ cell tumor with
trophoblastic differentiation
• Caveat: CCs after Rx may lose syncytiotrophoblasts
and show scant to absent reactivity for βhCG
• Overview: Useful for supporting Dx of CC but
usually not necessary
Useful Antibodies for Testicular Neoplasms:
Placental Alkaline Phosphatase (PLAP)
• An allosteric enzyme in placental trophoblast
• + in IGCNU (83-100%) & <1% of non-neoplastic
germ cells
• + in 90-100% of usual seminomas with a
membranous pattern. Most ECs & ~ 50% of YSTs &
CCs are +
• SS & SCT are negative
• Other + tumors: many adenocas (ovary, colon,
endometrium, lung)
• Caveat: Not a specific GCT marker
• Overview: Mostly helpful for IGCNU; useful for
usual seminoma vs SS or SCT
Immunohistochemistry Algorithm #1 for Testicular Neoplasia:
Germ Cell Tumor Subtyping
Germ Cell Tumor
– OCT 4
+ OCT4
+CD117
(Podoplanin,
SOX17)
– CD30
(AE1/AE3, SOX2)
– CD117
(Podoplanin,
SOX17)
+ CD30
(AE1/AE3, SOX2)
+Glypican 3
+ AFP
– hCG
+PLAP
– Glypican 3
– AFP
– hCG
– PLAP
+Glypican 3
– AFP
+ hCG
+ PLAP
Seminoma
Embryonal
Carcinoma
Yolk Sac
Tumor
Spermatocytic
Semimoma
Choriocarcinoma
Preferred markers in bold; alternatives in parentheses
Proposed ISUP Recommendations:
Germ Cell Tumor Subtyping
• A reasonable and efficient initial panel
is: OCT4, CD117, CD30 & GPC3
• This panel may be reduced depending
on the light microscopic differential, for
instance omitting OCT4 & GPC3 if the
question by morphology is limited to
seminoma versus embryonal
carcinoma
Solid YST with clear cells, potentially mimicking seminoma
Solid YST – OCT4
Seminoma – OCT4
Solid YST – glypican 3
Seminoma & EC
Seminoma & EC – CD30
Seminoma & EC – CD117
Useful Antibodies for Testicular Neoplasms:
SALL4
• Zinc finger nuclear transcription factor with role in
embryonic development
• ~100% sensitive for IGCNU, seminoma, EC & YST;
69% of CCs & 52% of teratomas
• Negative in other testis tumors
• Other + tumors: ALCL, rhabdoid tumor, Wilms
tumor, precursor B-cell ALL, AML & ~ 5% of GI
tract adenoca
• Caveat: Non-neoplastic germ cells are +
• Overview: Sensitive general GCT marker valuable
for GCT vs non-GCT of testis and in DX of
metastatic GCTs; sensitive YST marker, unlike
OCT4
Useful Antibodies for Testicular Neoplasms:
Inhibin
• A dimeric glycoprotein in the transforming growth
factor-ß superfamily. Inhibits production or secretion
of pituitary gonadotropins. Ab vs the α subunit is used
• Positive in sex cord-stromal tumors (SCST); ~100% of
Leydig cell tumors (LCT) & 30-91% SCTs
• Positive in syncytiotrophoblast cells but GCTs are
otherwise negative
• Other + tumors: any with trophoblast cells, adrenal
cortical, hemangioblastoma, some CCRCCs, rare soft
tissue
• Caveat: Negative α-inhibin does not exclude SCST
• Overview: Helpful positive stain for SCST vs GCT
Useful Antibodies for Testicular Neoplasms:
Calretinin
• A 29 kDa calcium-binding protein of the E-F hand
protein family.
• Positive in SCSTs, including ~ 100% of LCTs but
only a minority of SCTs
• GCTs are negative
• Other + tumors: mesothelioma, adenomatoid
tumors and many others
• Caveat: Negative calretinin does not exclude
SCST
• Overview: Helpful positive stain for SCST vs
GCT
Immunohistochemistry Algorithm #2a for Testicular Neoplasia:
Germ Cell Tumor vs Sex Cord-Stromal Tumor
Germ Cell Tumor
vs
Sex Cord-Stromal
Tumor
+ SALL4
– Inhibin & Calretinin
– SALL4
+ or – Inhibin &
Calretinin
Germ Cell Tumor
(see Algorithm 1)
Sex Cord-Stromal
Tumor
Immunohistochemistry Algorithm #2b* for Testicular Neoplasia:
Germ Cell Tumor vs Sex Cord-Stromal Tumor
Germ Cell Tumor
vs
Sex Cord–
Stromal Tumor
– OCT4
+ OCT4
Embryonal
Carcinoma
or
Seminoma
(see Algorithm
1)
* Alternative algorithm if
SALL4 not available
+ Glypican 3
± AFP
± PLAP
– Inhibin & Calretinin
– Glypican 3
– AFP
– PLAP
± Inhibin & Calretinin
Yolk Sac
Tumor
Sex Cord–
Stromal
Tumor
Proposed ISUP Recommendations:
Germ Cell Tumor versus Sex CordStromal Tumor
• A reasonable and efficient initial panel
is: SALL4, Inhibin & Calretinin
• An alternative panel is OCT4, GPC3,
Inhibin & Calretinin
Immunohistochemistry Algorithm #3a for Testicular
Neoplasia: Germ Cell Tumor vs Large Cell Lymphoma
Germ Cell Tumor
vs
Large Cell Lymphoma
+ SALL4*
– SALL4*
Germ cell tumor
(see Algorithm 1 for
subtyping)
+ Specific lymphoid
markers
(CD45, CD20, PAX5,
CD79a, CD3, others)
Large Cell
Lymphoma
* Some lymphoblastic & ALCLs & myeloid leukemias are SALL4 +.
Immunohistochemistry Algorithm #3b* for Testicular Neoplasia:
Germ Cell Tumor vs Large Cell Lymphoma
Germ Cell Tumor
vs
Large Cell Lymphoma
–
OCT4†
+
OCT4†
Embyronal
Carcinoma
or
Seminoma
(see Algorithm 1)
+ Glypican 3
+ AE1/AE3
– Glypican 3
– AE1/AE3
+ Specific lymphoid
markers (CD45, CD20,
PAX5, CD79a, CD3, others)
Yolk Sac
Tumor
Large Cell
Lymphoma
* Alternative algorithm if SALL4 not available; †rare large cell lymphomas are
OCT4 +
Proposed ISUP Recommendations:
Germ Cell Tumor versus Large Cell
Lymphoma
• A reasonable and efficient initial panel
is: SALL4, CD45, CD20 & CD3
• An alternative initial panel is OCT4,
GPC3, AE1/AE3, CD45, CD20 & CD3
Useful Antibodies for Testicular Neoplasms:
Epithelial Membrane Antigen (EMA)
• Glycoprotein in human milk fat globule
membranes
• Positive in most carcinomas and some
sarcomas & lymphomas (ALCL)
• Rarely positive in seminomas (2%), YSTs
(2%) & ECs (2-12%)
• Caveat: Negative GCT marker; not entirely
specific in DX of GCT vs non-GCT
• Overview: Useful in the differential of GCT
versus somatic carcinoma.
Useful Antibodies for Testicular Neoplasms:
Cytokeratin 7 (CK7)
• Type II keratin of simple nonkeratinizing
epithelium
• Positive in many carcinomas
• Negative in YST
• May be positive in non-YST GCTs
• Caveat: A negative YST marker & some
carcinomas are negative (prostate, colon,
etc.)
• Overview: Mostly useful in the differential of
YST vs somatic carcinoma
Immunohistochemistry Algorithm #4a for Testicular Neoplasia:
Germ Cell Tumor vs Metastatic High Grade Carcinoma
Germ Cell Tumor
vs
Metastatic High Grade
Carcinoma
+ SALL4
+ OCT4*
– EMA
– SALL4 (rarely +)
– OCT4
+ EMA
Germ Cell Tumor
(see Algorithm 1)
Metastatic High Grade
Carcinoma
*Embryonal carcinoma and Seminoma only
Immunohistochemistry Algorithm #4b* for Testicular Neoplasia:
Germ Cell Tumor vs Metastatic High Grade Carcinoma
Germ Cell Tumor
vs
Metastatic High Grade Carcinoma
+ OCT4
– EMA
Seminoma
or
Embryonal
Carcinoma
(see Algorithm 1)
– OCT4
+ Glypican 3
– EMA
– Cytokeratin 7
– Glypican 3†
+ EMA
± Cytokeratin 7
Yolk Sac Tumor
High Grade
Carcinoma
* Alternative algorithm if SALL4 not available
† Hepatocellular carcinomas, hepatoid carcinomas of other organs and squamous cell carcinomas
may be glypican 3 positive.
Proposed ISUP Recommendations:
Germ Cell Tumor versus Metastatic High
Grade Carcinoma
• A reasonable and efficient initial panel
is: SALL4, OCT4 & EMA
• An alternative panel is OCT4, GPC3,
EMA & CK7
Immunohistochemistry Algorithm #5 for Testicular Neoplasia:
Seminoma with Syncytiotrophoblasts vs Choriocarcinoma
Preferred markers in bold; alternatives in parentheses
Seminoma+SynT
Seminoma+SynT – OCT4
Immunohistochemistry Algorithm #6 for Testicular Neoplasia: Intratubular Germ
Cell Neoplasia Unclassified (IGCNU) vs Non-neoplastic Atypical Germ Cells
IGCNU
vs
Nonneoplastic Atypical
Germ Cells
+ OCT4
(PLAP, Podoplanin)
( not SALL4, SOX17 or
CD117)
– OCT4
(PLAP, Podoplanin)
(not SALL4, SOX17 or
CD117)
IGCNU
(rule out maturation
arrest in young
children)
Nonneoplastic Atypical
Germ Cells
Preferred marker in bold