1 Diagnostic Antibody Panel for Tumors of the Vulva and Vagina

Cytokeratin profile, p63, CEA, p16, HPV, steroid hormone receptors, desmin, myogenin, and melanoma markers.

2 Diagnostic Antibody Panel for Tumors of the Uterine Cervix

Cytokeratin profile, p63, CEA, PAX-8, PAX-2, p16, p53, HPV, and steroid hormone receptors.

3 Diagnostic Antibody Panel for Epithelial Tumors of the Uterine Corpus, Fallopian Tube, and Uterine Ligament

Cytokeratin profile, CEA, PAX-8, p16, p53, HNF-1β, and steroid hormone receptors.

4 Diagnostic Antibody Panel for Uterine Mesenchymal Tumors

Smooth muscle markers, CD10, and steroid hormone receptors.

p16

Expression pattern: nuclear/cytoplasmic

Main diagnostic use

Expression in other tumors

Expression in normal cells

HPV-associated oropharynx and uterine cervix squamous cell carcinoma, atypical lipomatous tumors and liposarcoma

Endometrial serous carcinoma, clear cell carcinoma, melanocytic nevi and melanoma, adenoid cystic carcinoma, malignant mesenchymal tumors

 

Positive control: cervical squamous cell carcinoma

Diagnostic Approach

P16 (also known as INK4a or cyclin-dependent kinase inhibitor 2A) is a tumor suppressor protein encoded by the p16INK4a gene. p16 inhibits the cyclin-dependent kinases [1, 2] involved in in cell cycle regulation and progression (G1 to S). p16 plays role in the pathogenesis of different malignancies. The expression of p16 is regulated by the retinoblastoma (Rb) gene, which in turn is affected by the E7 oncogene of the HPV gene. p16 is overexpressed in HPV-associated intraepithelial dysplasia and squamous cell carcinomas of different origins including vulvar, vaginal, and cervical squamous cell carcinoma in addition to oropharynx carcinoma. In routine immunohistochemistry, p16 reveals cytoplasmic and nuclear staining pattern and the intensity of the stain correlates with grade of HPV infection and grade of associated dysplasia. p16 is also highly expressed in uterine serous carcinoma and a helpful marker that labels the cells of serous tubal intraepithelial carcinoma (STIC) [3].

p16 is also a useful marker to discriminate between atypical lipomatous tumors (well-differentiated liposarcoma) or other liposarcoma types positive for p16 and benign adipocytic tumors lacking the expression of p16 [4, 5].

PAX-8:

PAX-8 is a transcriptional factor involved in the fetal development of the brain, eye, thyroid tissue, kidney, and upper urinary system as well as the Müllerian organs. PAX-8 is listed in detail in a next chapter.

Hepatocyte Nuclear Factor-1β (HNF-1β):

HNF-1β is a member of the hepatocyte nuclear factor family regulating the growth and differentiation of hepatocytes and cells of the biliary system. The expression of different hepatocyte nuclear factors is not restricted to the liver but variously found in other organs including the pancreas, kidney, prostate, and female genital system [6]. HNF-1β is used in diagnostic immunohistochemistry to differentiate between different types of ovarian and endometrial carcinomas. The strong nuclear HNF-1β expression is characteristic for both endometrial and ovarian clear cell carcinomas but usually negative in reactive lesions with clear cell appearance such as clear cell metaplasia and Arias-Stella phenomenon [7]. However, we must consider that focal weak to moderate HNF-1β expression can be also found in other endometrial and ovarian carcinoma types such as endometrioid and serous carcinomas [8]. Additionally, different HNF-1β expression intensity is also found in other carcinomas of different origin including colorectal, pancreatobiliary, prostatic, and renal cell carcinomas.

Phosphatase and Tensin Homolog (PTEN):

PTEN is a widely expressed enzyme in mammalian cells that catalyzes the dephosporylation of the 3` phosphate of the inositol ring, an essential reaction that causes the inhibition of the protein kinase (AKT) signaling pathway involved in the regulation of apoptosis. Mutations that inactivate the PTEN gene cause the inhibition of the apoptotic cascade increasing cell proliferation. Inactivating mutations within the PTEN are commonly seen in different human neoplasias such as urogenital, breast, and lung carcinomas in addition to melanoma and glial tumors [9]. The immunohistochemical staining of PTEN (cytoplasmic pattern) is a simple way to detect the loss of this enzyme. The loss of PTEN expression is found in 30–50% of endometrial carcinoma and in about 25% of endometrium with atypical complex hyperplasia, which indicates that the loss of PTEN is not a specific marker of malignant transformation [10, 11]. Normal proliferative endometrium shows usually strong PTEN expression. The loss of PTEN expression is also found in a subset of ovarian endometrioid carcinoma (~20%), high-grade serous carcinoma, and clear cell carcinoma.

A fraction of high Gleason prostatic carcinoma is also associated with PTEN loss (see markers of prostatic carcinoma) [9]. PTEN mutations are found in primary glioblastoma but rare in secondary glioblastoma.

Steroid Receptors:

Both estrogen and progesterone receptors were discussed in details with the markers of breast tumors. Endometrial adenocarcinoma and serous endometrial carcinoma are sex hormone-dependent tumors, and the expression of estrogen and progesterone is characteristic for both carcinoma types [12]. Myometrium is also a target tissue for steroid hormones; accordingly the majority of uterine leiomyomas and leiomyosarcomas are positive for estrogen receptors, progesterone receptors, or both. This characteristic feature can be used to differentiate between uterine and soft tissue leiomyosarcoma [13]. Squamous cell carcinoma and adenocarcinoma of uterine cervix usually lack the expression of both receptors [14].

Immunoprofile of tumors of the uterine cervix, uterine corpus, and fallopian tube

Tumor type

+ in >90% (+)

+ in 50–90% (±)

+ in 10–50% (∓)

+ in <10% (−)

A. Tumors of the vulva and vagina

Paget’s disease of the vulva

CK7, EMA (MUC1), CEA, androgen receptors

ER

GCFP-15

CK5/6/14, CK20

Squamous cell carcinoma

CK5, CK6, CK18, CK19, P16

  

CK7, CK20

Bartholin gland carcinoma

• Adenocarcinoma

• Squamous cell carcinoma

• Adenoid cystic carcinoma

• Transitional cell carcinoma

See immunoprofile of similar carcinomas of other locations

Adenocarcinoma of mammary type

See immunoprofile of breast carcinoma

Adenocarcinoma of Skene gland type

Pan-CK, PSA

  

PAX-8

Clear cell carcinoma

CK7, EMA, CEA

  

CK20

Sebaceous carcinoma

Adipophilin, EMA, androgen receptors

Perilipin, CK5/14, CK8/18, CK7, CK19, CD15, p16

 

CK20, CEA, S100

Angiomyofibroblastoma

Desmin

ER, PgR

CD34

Actin

Cellular angiofibroma

 

CD34, ER, PgR

Actin

 

Superficial angiomyxoma

CD34

  

Actin, desmin, S100

Deep aggressive angiomyxoma

Desmin, HMGA2

Actin, ER, PgR

CD34, actin, S100

Myogenin, MyoD1

Epithelioid sarcoma

See miscellaneous soft tissue tumors

Rhabdomyosarcoma

See soft tissue rhabdomyosarcoma

B. Tumors of the uterine cervix

Squamous cell carcinoma of the cervix and uterus

CK5, CK6, CK13, CK17, CK18, CK19, P16

CK14

 

CK7, CK20, ER, PgR

Endocervical adenocarcinoma

CK7, CK8, CK18, CK19, CEA, EMA, p16, PAX-8

 

CK20, vimentin

ER, PgR, CK5/6, WT-1, PAX-2a, GFAP

Endometrioid adenocarcinoma

CK7, CK8, CK18, CK19, EMA

ER, PgR, vimentin, GFAP

p16, CD56

CK20, CK5/6, CEA, CDX-2

Mesonephric adenocarcinoma

CK5/6, CK7, CK8, CK18, EMA, CD15

CD10, p16, calretinin, vimentin, bcl-2

Androgen receptors, PAX-8, TTF-1

ER, PgR, CK20, CEA

Adenosquamous carcinoma/glassy cell carcinoma

CK7b, CK5/6/14c

  

ER, PgR

Adenoid basal carcinoma

CK5/14, p63, p16

   

Neuroendocrine tumors

• NET(c) G1

• NET(d) G2

• NEC(e) G3 (small cell carcinoma)j, k, l

Pan-CK, CD56, NSE, PGP9.5

Proliferation index (Ki-67) in

NET G1: <2%

NET G2: 3–20%

NEC G3: >20%

Synaptophysin, chromogranin

TTF-1

CK7, CK20

C. Tumors of the uterine corpus

Endometrial adenocarcinoma

CK7, CK8, CK18, CK19, PAX-8, EMA, CA125

PgR, ER, vimentin, GFAP

CD56, p53, P16

CK20, CK5/6, CEA, WT-1, IMP3, CDX-2d

Serous endometrial carcinoma

CK7, CK8, CK18, CK19, EMA, CA125, p16, p53, PAX-8, β catenin

Proliferation index (Ki-67): >75%

IMP3, PgR, ER

ER, PgR, Sox-2, WT-1

CK5/6, CK20, HNF1-β

Clear cell carcinoma

CK 7, EMA, CA125, PAX-8, hepatocyte nuclear factor 1-β (HNF1-β), p504s (AMACR)

Vimentin, CD15

ER, AFP, CEA, p16, p53, Sox-2

PgR, WT-1, CK20, CD10

Undifferentiated carcinoma

EMA, vimentin

Pan-Cytokeratin, CK8/18, p53

PAX-8, synaptophysin, chromogranin

ER, PgR

Low-grade endometrial stromal sarcoma

CD10, β-catenin, vimentin

ERα, PgR, bcl-2, WT-1, TLE-1

Cyclin D1, androgen receptors, actin, desmin, pan-CK

h-Caldesmon, calponin, CD34, EMA, inhibin, oxytocin receptor

High-grade endometrial stromal sarcoma

Cyclin D1

CD117

 

CD10, ER, PgR

Uterine leiomyoma/leiomyosarcoma

Desmin, actin, calponin, oxytocin receptor, p16e, p53e, vimentin

Proliferation index (Ki-67) in uterine leiomyoma: <5%

Proliferation index (Ki-67) in atypical uterine smooth muscle tumors: 5–10%

Proliferation index (Ki-67) in uterine leiomyosarcoma: >15%

h-Caldesmon, ER, PgR

Pan-CK

CD10, EMA

Perivascular epithelioid tumor of the uterus (PEComa)

HMB45, Melan A, tyrosinase, MITFf, CD63 (NK1-C3)

 

Actin, desmin

CD10, CD34, pan-CK, S100

Placental site trophoblastic tumor

Human placental lactogen, CD146, inhibin, pan-CK

Proliferation index (Ki-67): >10%g

 

ßhcG

 

Gestational choriocarcinoma

See choriocarcinoma of the ovary

  

D. Tumors of the fallopian tube

Serous tubal intraepithelial carcinoma (STIC)h

p53, p16, stathmin 1i

Ki-67 > 15%

   

Serous carcinoma

CK7, CK8, CK18, CK19, EMA, WT-1, p53, p16

ER, PgR

 

CK5/6, CK20

Endometrioid adenocarcinoma

CK7, CK8, CK18, CK19, EMA, ER

PgR, GFAP, vimentin

p53, CD56

P16, CK20, CK5/6, CEA, CDX-2

Undifferentiated carcinoma

EMA, vimentin

Pan-cytokeratin, CK8/18

Synaptophysin, chromogranin

ER, PgR

E. Tumors of uterine ligaments

Epithelial tumors of Müllerian type

See uterine tumors

  1. aPAX-2 is usually expressed in benign proliferating endocervical glands
  2. bCK7 positive in glandular components
  3. cCK5/6/14 positive in squamous components
  4. dCDX-2 may be positive in mucinous-type endometrioid adenocarcinoma
  5. eP16 and p53 usually positive only in leiomyosarcoma
  6. fMicrophthalmia transcription factor
  7. gProliferation index (Ki-67) in placental site nodule and exaggerated placental site <1% and >50% in choriocarcinoma
  8. hSee Fig. 11.1
  9. iDiffuse expression in STIC lesions but few scattered cells in normal fallopian mucosa [3]
  10. jWell-differentiated neuroendocrine tumor (carcinoid)
  11. kWell-differentiated neuroendocrine tumor (atypical carcinoid)
  12. lPoorly differentiated neuroendocrine carcinoma
Fig. 11.1
figure 1

Serous tubal intraepithelial carcinoma (STIC). (a, b) H&E 40X and 200X showing the fallopian tube with marked atypia of tubal epithelium, (c) same section with strong diffuse nuclear p53 expression, (d) Ki-67 expression in ~15% of epithelial cells

Full size image

5 Tumors of the Ovary

5.1 Diagnostic Antibody Panel for Ovarian Epithelial Tumors

Cytokeratin profile, CEA, CA125, PAX-8, WT-1, p53, p16, GATA-3, S100P, steroid hormone receptors, and HNF-1β.

5.2 Diagnostic Antibody Panel for Ovarian Germ Cell Tumors

CD117, PLAP, Oct-4, SALL-4, Sox-2, AFP, CD30, βhcG, and cytokeratin profile (see also testicular germ cell tumors).

5.3 Diagnostic Antibody Panel for Ovarian Sex Cord-Stromal Tumors

Inhibin, anti-Müllerian hormone, FOXL-2, Melan A, CD56, CD99 (see also testicular sex cord-stroma tumors).

Wilms’ tumor protein-1 (WT-1)

Expression pattern: nuclear

Main diagnostic use

Expression in other tumors

Expression in normal cells

Nephroblastoma, mesothelioma, malignant melanoma, metanephric adenoma, ovarian serous carcinoma, carcinoma of the fallopian tube

Acute myeloid leukemia, Burkitt lymphoma and subset of ALL, desmoplastic small round cell tumor, endometrial stromal sarcoma, uterine leiomyosarcoma, sex cord-stromal tumors (granulosa cell tumor, fibroma, fibrothecoma, Sertoli cell tumor), Brenner tumor, ovarian small cell carcinoma of hypercalcemic type, neuroblastoma, rhabdoid tumor, rhabdomyosarcoma

Renal tissue (glomerular podocytes), mesothelial cells, granulosa cells, Sertoli cells, fallopian tube, endometrial stroma, spleen, breast tissue, bone marrow stem cells

Positive control: appendix

Diagnostic Approach

Wilms’ tumor protein-1 (WT-1) is a transcriptional regulator encoded by the WT-1 gene on chromosome 11p13 with four isoforms. WT-1 plays an important role in the regulation of growth factors and development of tissues from the inner layer of intermediate mesoderm including the genitourinary system, mesothelial cells, and spleen. Mutation within the WT-1 gene affecting the DNA-binding domain can cause the development of nephroblastoma. In routine immunohistochemistry, WT-1 shows two different expression patterns: first, a true nuclear expression pattern characteristic for different tumors such as serous carcinomas of ovarian, tubal, and peritoneal origin and mesothelioma (Fig. 11.2); secondly a cytoplasmic staining pattern found in endothelium and vascular tumors in addition to some carcinoma types such as pulmonary adenocarcinoma [1]. The cytoplasmic expression pattern appears to result from a cross reactivity with other epitopes unrelated to the WT-1 transcription factor. Endometrioid, clear cell, transitional, and mucinous carcinomas are usually WT-1 negative or show focal weak positivity. WT-1 is a helpful marker to differentiate between WT-1 positive tumors and many other WT-1 negative tumors with similar morphology such as neuroblastoma and the PNET tumor group.

Fig. 11.2
figure 2

Serous ovarian carcinoma with strong nuclear WT-1 expression

Full size image

Diagnostic Pitfalls

WT-1 labels a high percentage of epithelioid mesotheliomas, which to consider in the differential diagnosis between ovarian peritoneal carcinosis and primary peritoneal mesotheliomas. For differential diagnosis, other antibodies such as PAX-8, Ber-EP4, and calretinin are helpful.

CA125 (MUC-16)

Expression pattern: membranous (luminal surface)

Main diagnostic use

Expression in other tumors

Expression in normal cells

Ovarian carcinoma (serous, endometrioid and clear cell carcinomas)

Lung, breast, gastrointestinal, uterine, and seminal vesicle adenocarcinomas, yolk sac tumor, epithelioid mesothelioma, anaplastic large cell lymphoma, desmoplastic small round cell tumor

Breast ductal epithelium, epithelium of the lung, gastrointestinal tract, biliary system, pancreas, female genital tract and apocrine glands, mesothelial cells

Positive control: serous ovarian carcinoma

Diagnostic Approach

Carbohydrate antigen 125 (CA125) is a high molecular weight glycoprotein classified as mucin 16 (MUC-16). CA125 is normally expressed by glandular epithelium of different organs and is highly expressed in ovarian serous and clear cell carcinomas (Fig. 11.3). Serum CA125 is also used to monitor the progression of ovarian carcinoma.

Fig. 11.3
figure 3

Serous ovarian carcinoma with membranous CA125 expression

Full size image

Diagnostic Pitfall

CA125 is expressed by different epithelial and non-epithelial malignancies and lacks the specificity to ovarian carcinoma. Mesotheliomas can also be positive to CA125.

PAX-8:

PAX-8 is a transcriptional factor and a member of the paired box (PAX) family listed in detail with the markers of renal cell tumors. PAX-8 is highly expressed in Müllerian glandular epithelia as well as in renal tubules and upper urinary system. PAX-8 strongly labels all uterine, endocervical, and ovarian tumors of Müllerian origin including serous, clear cell, and endometrioid carcinomas.

Hepatocyte Nuclear Factor-1β (HNF-1β):

See the previous chapter (Chap. 10).

FOXL2

Expression pattern: nuclear

Main diagnostic use

Expression in other tumors

Expression in normal cells

Sex cord-stromal tumors

Breast cancer, pituitary gland adenoma

Granulosa cells, subset of pituitary cells

Positive control: ovarian tissue (granulosa cells)

Diagnostic Approach

FOXL2 (forkhead box transcription factor L2) is a transcriptional factor involved in the development of the ovaries and female genital tract. FOXL2 is highly expressed in testicular and ovarian sex cord-stromal tumors including adult and juvenile granulosa cell tumors, thecoma/fibroma, Sertoli/Leydig cell tumors and sclerosing stromal tumor. Subset of pituitary gland adenomas is also positive for FOXL2, namely, gonadotropins producing adenomas and majority of null cell adenomas [2, 15, 16]. Ovarian surface epithelial tumors and germ cell tumor are FOXL2 negative.

Immunoprofile of ovarian tumors

Tumor type

+ in >90% (+)

+ in 50–90% (±)

+ in 10–50% (∓)

+ in <10% (−)

A. Ovarian epithelial tumors

Serous ovarian neoplasms

• Adenoma

• Borderline

• Low-grade carcinoma

• High-grade carcinoma

CK7, CK8, CK18, CK19, EMA, CA125, WT-1, PAX-8, p53 a, p16 a, HAM56

Median proliferation index (Ki-67) in serous carcinoma:

Low grade ~ 2,5%

High grade ~ 22%

CK5/6, mesothelin

Vimentin, ER, PgR, calretinin, S100, TTF-1, CD99

Villin, CK20, CEA, MUC-2, CDX-2, inhibin

Mucinous ovarian neoplasms (adenoma, borderline, and carcinoma)

CK7, CK8, CK18, CK19, EMA

CK20b, CDX-2b, MUC-2, MUC5AC, CEA, PAX-8, p53c

Villin

WT-1, p16, ER, PgR, CK17, vimentin, inhibin, TTF-1

Endometrioid carcinoma

CK7, CK8, CK18, CK19, EMA, PAX-8, ER, CA125

Vimentin, mesothelin, CD99

WT-1, p16, CK5

CK20, WT-1, CEA, inhibin, TTF-1

Clear cell adenocarcinoma

Hepatocyte nuclear factor 1-β (HNF1-β), PAX-8, CK7, EMA

Vimentin, CD15, CA125

AFP, CEA, napsin A, p53

WT-1, p16, ER, PgR, CK20, CD10

Brenner tumor (benign/malignant)

Epithelial components: EMA, CK7, p63, CEA, CK5/6/14 d, CA125, Uroplakin III

Fibrous stroma: vimentin

WT-1, S100P, PAX-8

bcl-2

 

CK19, CK20, thrombomodulin (CD141), vimentin

Pan-CK

B. Sex cord-stromal tumors

Granulosa cell tumor

FOXL2, adrenal 4 binding protein (SF-1), inhibin, vimentin

Calretinin, CD99, actin, S100, CD56, WT-1, ERβ, PgR

Pan-CK, CK8, CK18, ERγ

CK7, EMA, CEA, anti-Müllerian hormone, desmin

Thecoma /Fibroma

Inhibin, FOXL2, adrenal 4 binding protein (SF-1), WT-1, calretinin, vimentin

sm-actin

ER, PgR

Pan-CK

Sclerosing stromal tumor

sm-Actin, PgR, FOXL2, vimentin

Inhibin, calretinin, desmin

ER

Pan-CK

Leydig cell tumor

Inhibin, Melan A, calretinin, vimentin

CD99, CD56

Pan-CK, S100, actin, desmin, synaptophysin, chromogranin, EMA

PLAP, AFP, CEA

Sertoli cell tumor

Inhibin, adrenal 4 binding protein (SF-1), FOXL2, anti-Müllerian hormone, WT-1, Melan A, vimentin

AFP, CD56, CD99, pan-CK, calretinin, NSE, S100

Synaptophysin, chromogranin

EMA, PLAP, CEA

Sex cord tumor with annular tubules

Inhibin, adrenal 4 binding protein (SF-1), WT-1, calretinin

CD56

Pan-CK

EMA

C. Germ cell tumors

Dysgerminoma

SAL4, Oct-4, NANOG, PLAP, CD117

Pan-CK, D2-40

CK8/18

AFP, ßhcG, Sox-2, inhibin, S100, EMA

Embryonal carcinoma

SALL-4, NANOG, Sox-2, PLAP, AFP, CD30, Oct-4, pan-CK

CK19, NSE

 

ßhcG, EMA, CEA, CD117, vimentin

Yolk sac tumor

AFP, SALL-4, pan-CK, CD10, glypican-3

PLAP

CDX2, HepPar1

EMA, CD30, ßhcG, Oct-4, Sox-2, CK7, vimentin

Choriocarcinoma

Syncytiotrophoblastic cells: ßhcG, inhibin, CD10, pan-CK, CK8/18, CK19, GATA-3, EGFR

Cytotrophoblastic cells: CD10, pan-CK, CK8/18, CK19, CEA

PLAP, human placental lactogen, EMA, CEA

PLAP

Vimentin

CD30, AFP,

Oct-4

ßhcG, inhibin, EMA, CD30, AFP, Oct-4

Polyembryoma

In embryonal bodies: AFP, pan-CK

PLAP

  

Gonadoblastoma

Germ cells: PLAP, CD117, Oct-4, NANOG, D2-40

Sex cord cells: inhibin, WT-1, vimentin

Pan-CK

  

D. Miscellaneous tumors

Female adnexal tumor of probable Wolffian origin (ovarian Wolffian tumor)

Pan-CK, CK7, androgen receptors, vimentin

Calretinin, CD10, Melan A

Inhibin

EMA, CK5/6, CK20, CEA

Small cell carcinoma, hypercalcemic type

EMA, WT-1

Calretinin, CD56

Synaptophysin, chromogranin

CD10, inhibin

Small cell carcinoma, pulmonary type

NSE, CD56

TTF-1

Synaptophysin, chromogranin

 
  1. aHigh expression level characteristic for high-grade serous carcinoma, low expression level or negative in low-grade carcinoma
  2. bCDX-2 and CK20 positive in mucinous adenocarcinoma and intestinal type adenoma
  3. cUsually negative in adenoma and borderline tumors
  4. dCK5/6/14 positive in basal epithelial cells