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Protein Expression Education Series - Breast Module

“This presentation was prepared by Philip Ferguson M.D., a board certified pathologist practicing in Jonesboro, AR, USA. Dr. Ferguson was compensated for this presentation. The presentation represents Dr. Ferguson’s views, and does not necessarily represent or reflect the views or opinions of Leica. It is for educational purposes only and is not intended as regulatory or legal advice or as diagnostic advice for a particular patient or a particular disease or condition.”


CK7

 

CK7 is an intermediate filament with a selective expression pattern in different carcinomas, which when combined with CK20 is useful in the work up of carcinomas of unknown primary origin.

CK7 is part of a group of cytokeratins often referred to as low-molecular weight keratins (LMWK). In general these cytokeratins (CK7, CK20 CK8, CK18, and CK19) are expressed in simple epithelium (as opposed to high molecular weight cytokeratins expressed in stratified epithelium). The distribution of expression of these LMWK is dependent upon the organ system. This differential expression can be helpful when narrowing a differential diagnosis for a carcinoma of unknown primary site (CUPS). Most commonly CK7 is combined with CK20 in a panel, which dependent upon the differential expression pattern, more specific markers can be added to narrow the diagnostic possibilities (see CK7/CK20 tables below).

Common expression patterns in carcinoma (Dennis, JL, et al).

Tumor

Expression%

Breast >80%
Colon<10%
Lung>90%
Ovary>80%
Pancreas >80%
Stomach 35-70%
Prostate ~10%

 

Cytokeratin expression in various tumors (Moll, RT, et al. )

Tumor

CK8/CK18

CK19

CK7

CK20

CK5

Hepatocellular Ca. + ++/-+=
Colorectal ACA+ ++/-+=
Stomach ACA+ ++/-+/-=
Pancreas Ductal ACA+ +++/-+/-
Lung ACA+ ++==
Breast Inv. Ductal+ ++=+/-
Endometrium ACA+ ++=+/-
Ovary ACA+ ++==
RCC, Clear Cell Type+ +/-===
RCC, Papillary Type+ ++==
RCC, Chromophobe+ +/-++=
Mesothelioma+ ++/-=+
Lung, Small Cell Ca.+ +/-===
Merkel Cell Ca.+ +=+=
Urothelial Carcinoma+ +++/-+/-
Squamous Cell Ca.+/- +/-==+


Key: "+/-“, focal staining in some cases. “=“, negative, “+”, positive.

 

CK7 - Breast

CK7 expression in skin highlighting duct epithelium of eccrine/adnexal structures. CK7 staining pattern is the inverse of CK5 expression.

CK7 – Breast

CK7 expression in eccrine/adnexal structures from a skin section. CK7 with stain in an “inverse” pattern with epithelium compared to CK5.

CK7 – Breast

CK7 expression in lung adenocarcinoma

 

References:

Hadi, AIMM Annual Meeting, "The Thirty Most Important Antibodies", presentation, 2011.

Dennis, J. L., Hvidsten, T. R., Wit, E. C., Komorowski, J., Bell, A. K., Downie, I., et al. (2005). Markers of adenocarcinoma characteristic of the site of origin: development of a diagnostic algorithm. Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, 11(10), 3766–3772. doi:10.1158/1078-0432.CCR-04-2236

Moll, R., Divo, M., & Langbein, L. (2008). The human keratins: biology and pathology. Histochemistry and Cell Biology, 129(6), 705–733. doi:10.1007/s00418-008-0435-6

Moll R. Cytokeratins as markers of differentiation. Expression profiles in epithelia and epithelial tumors. Veroffentlichungen aus der Pathologie. 1992.

 

CK20

CK20 is an intermediate filament with a selective expression pattern in different carcinomas, which when combined with CK7 is useful in the work up of carcinomas of unknown primary origin. CK20 has also been used in combination with other markers to evaluate potentially neoplastic urothelium based on proliferation and epithelial maturation characteristics. In other settings, loss of normal expression (e.g. CK20/CDX-2 expression loss in colon adenocarcinoma) has been associated with prognostic and molecular characteristics (e.g. BRAF mutation).

Common expression patterns in carcinoma (Dennis, JL, et al).

Tumor

Postive(%)

Breast ><10%
Colon<>80%
Lung<10%
Ovary<10%
Pancreas 35-50%
Stomach 30-50%
Prostate <10%

Moll, RT, et al. Cytokeratin expression in various tumors.

Tumor

CK8/CK18

CK19

CK7

CK20

CK5

Hepatocellular Ca. + +/-+/-+/-=
Colorectal ACA+ ++/-+=
Stomach ACA+ ++/-+/-=
Pancreas Ductal ACA+ +++/-+/-
Lung ACA+ ++==
Breast Inv. Ductal+ ++=+/-
Endometrium ACA+ ++=+/-
Ovary ACA+ ++==
RCC, Clear Cell Type+ +/-===
RCC, Papillary Type+ ++==
RCC, Chromophobe+ +/-+==
Mesothelioma+ ++/-=+
Lung, Small Cell Ca.+ +/-===
Merkel Cell Ca.+ +=+=
Urothelial Carcinoma+ +++/-+/-
Squamous Cell Ca.+/- +/-==+

Key: "+/-“, focal staining in some cases. “=“, negative, “+”, positive.

 

CK20 – Breast

CK20 expression (or lack thereof) in lung adenocarcinoma (CK7+/CK20-).

CK20 – Breast

CK20 expression in colon adenocarcinoma.

CK20 – Breast

CK20 expression in underlying colon adenocarcinoma, and normal expression in overlying benign mucosa (increased expression as it becomes more superficial).

 

References:

Hadi, AIMM Annual Meeting, "The Thirty Most Important Antibodies", presentation, 2011.

Dennis, J. L., Hvidsten, T. R., Wit, E. C., Komorowski, J., Bell, A. K., Downie, I., et al. (2005). Markers of adenocarcinoma characteristic of the site of origin: development of a diagnostic algorithm. Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, 11(10), 3766–3772. doi:10.1158/1078-0432.CCR-04-2236

Moll, R., Divo, M., & Langbein, L. (2008). The human keratins: biology and pathology. Histochemistry and Cell Biology, 129(6), 705–733. doi:10.1007/s00418-008-0435-6

Wee A. Diagnostic utility of immunohistochemistry in hepatocellular carcinoma, its variants and their mimics. Appl Immunohistochem Mol Morphol. 2006;14: 266–272.

Yin H, He Q, Li T, Leong AS-Y. Cytokeratin 20 and Ki-67 to distinguish carcinoma in situ from flat non-neoplastic urothelium. Appl Immunohistochem Mol Morphol. 2006;14: 260–265.

Kim J-H, Rhee Y-Y, Bae JM, Cho N-Y, Kang GH. Loss of CDX2/CK20 expression is associated with poorly differentiated carcinoma, the CpG island methylator phenotype, and adverse prognosis in microsatellite-unstable colorectal cancer. Am J Surg Pathol. 2013;37: 1532–1541. doi:10.1097/PAS.0b013e31829ab1c1

 

CK7+ / CK20+

Tumor

CK7

CK20

TCC (Urothelial) ++
Ovarian Ca. (mucinous)+ +
Pancreatic Ca. (subset)++
Cholangiocarcinoma (subset)++
Gastric Adenoca. (subset) ++
Sm. Int. (non-Ampullary) Ca. ++ (60-70%)

Follow-up Markers for CK7+/CK20+

IHC Stain

Comment

Uroplakin Supportive of urothelial carcinoma
ThrombomodulinExpression may be seen in urothelial carcinoma or adenocarcinoma of the bladder
p63Expression supports urothelial carcinoma
CK5/6Expression supports urothelial carcinoma
CEA May be supportive of pancreatic ductal carcinoma
CA19-9 May be supportive of pancreatic ductal carcinoma
CDX-2 ~+/- expression seen in pancreatic ductal carcinoma, adenocarcinoma of the bladder, and ovarian mutinous carcinoma
GATA-3 Expression supports urothelial carcinoma

References

Arch Pathol Lab Med. Vol 134, February 2010.

Hadi, AIMM Annual Meeting, “Carcinomas of Unknown Primary”, presentation, 2011.

 

CK7+ / CK20-

Tumor

CK7

CK20

Breast Ca. (>95%) +- (0-16% are +)
Lung Adenoca. (90%)+ -
Ovarian Ca. (Serous) (>90%)+-
Pancreatic Ca. (Subset)+-
Mesothelioma +-
Endometrial Adenoca. +-
Lung Small Cell Ca. (60-80%) + (dot-like)-
Cholangiocarcinoma +-
Thyroid Carcinoma +-
Gastric Carcinoma (subset) +-


Follow-up Markers for CK7+/CK20-

TTF-1

Expression seen in lung adenocarcinoma, small cell carcinoma, and thyroid carcinoma (medullary, papillary and follicular)

MOC31 Expression commonly seen in lung adenoma, but not mesothelioma.
CK5/6Expression commonly seen in mesothelioma, but NOT lung adenoca.
ChromograninExpression supports neuroendocrine origin or differentiation (small cell carcinoma). Medullary thyroid ca. is also positive.
SynaptophysinExpression Supports neuroendocrine origin or differentiation (small cell carcinoma). Medullary thyroid ca. is also positive.
ER/PR Expression may be seen in breast carcinoma, ovarian serous carcinoma, and endometrial adenocaricnoma.
GCDFP15 Expression is fairly specific for breast carcinoma.
Mammaglobin Expression is fairly specific for breast carcinoma.
WT-1 Expression is often seen in mesothelioma and ovarian serous carcinomas.
CEA Expression may be seen in cholangiocarcinoma, breast carcinoma, and lung adenocarcinoma. Expression is NOT usually seen in ovarian serous carcinoma, endometrial adenocarcinoma, or mesothelioma.
Thyroglobulin Specific marker for follicular and papillary thyroid carcinoma. Medullary ca. does not express Thyroglobulin.
Calretinin Expression usually found in mesothelioma.
Calcitonin Expression is typical for medullary (thyroid) ca.


References

Arch Pathol Lab Med. Vol 134, February 2010.

Hadi, AIMM Annual Meeting, “Carcinomas of Unknown Primary”, presentation, 2011.

 

CK7- / CK20+

Tumor

CK7

CK20

Colon Adenoca. (75-95%) -+ (015% are -)
Merkel Cell Ca.- + (dot-like)


Follow-up Markers for CK7- / CK20+

IHC Stain

Comment

CDX-2 Expression supports colon origin
TTF-1Expression is not typically seen in Merkel cell ca.

CK20 negativity in colon adenocarcinoma may be due to incomplete fixation, or in some tumors CK20 and CDX-2 loss of expression may be associated with BRAF mutations.

 

References

Hadi, AIMM Annual Meeting, “Carcinomas of Unknown Primary”, presentation, 2011.

Arch Pathol Lab Med. Vol 134, February 2010.

 

CK7- / CK20-

Tumor

CK7

CK20

Hepatocellular Ca. - (9-17% are +)-
Renal Cell Ca.- -
Prostate Ca.-- (0-23% are +)
Neuroendocrine Ca.--
Squamous Cell Ca. -(+/-)-
Adrenal Cortical Ca. --
Gastric Adenoca. (subset) --


Follow-up Markers for CK7-/CK20-

IHC Stain

Comment

PSA Expression supports prostatic adenocarcinoma
PSAPExpression supports prostatic adenocarcinoma
CEACEA is NOT commonly expressed in prostate adencarcinoma, renal cell carcinoma, or adrenal cortical carcinoma.
CK5/6CK5/6 is NOT typically expressed in prostate adencarcinoma, which is in contrast to squamous cell carcinoma that usually shows expression.
VimentinExpression is commonly seen in renal cell carcinoma and adrenal cortical carcinoma.
RCC Ma.Commonly expressed in renal cell carcinoma.
EMAExpression commonly seen in renal cell carcinoma but not adrenal cortical carcinoma.
HepPar1Expression supports hepatocyte origin.
CD10RCC shows cytoplasmic expression, and a canalicular expression pattern is often seen in HCC.
pCEACanalicular patter is common in HCC
MOC-31HCC is negative for MOC-31. Cholangiocarcinoma (and many other carcinomas) are positive.
CK19Expression is NOT seen in HCC.
p63Expression is seen in squamous cell carcinoma.
InhibinExpression supports adrenal cortical origin
Melan-AExpression often seen in adrenal cortical carcinoma.


References

Arch Pathol Lab Med. Vol 134, February 2010.

Hadi, AIMM Annual Meeting, “Carcinomas of Unknown Primary”, presentation, 2011.

 

GATA-3

GATA-3 is a member of a subfamily of zinc finger transcription proteins, which has been found to be highly expressed in breast (>90%) (especially lunimal A breast carcinomas) and urothelial carcinomas (>80%).

The higher level of sensitivity compared to GCDFP-15 and mamaglobin (MGB), has made this marker useful differentiating primary breast carcinomas from other primaries. Some believe this marker has a better balance of sensitivity and specificity for breast carcinoma than GCDFP-15 and mamaglobin (MGB). Especially, in the setting of ER-negative tumors. However, specificity is variable dependent upon the differential diagnosis for a particular case (see tables below). GATA-3 use should be considered as part of IHC panels in the setting of carcinoma of unknown primary site. (Lin, et al)

GATA-3 may also have a role in the evaluation of possible primary bladder tumors. As with most IHC markers, there are very few “silver bullets,” and interpretation should take into consideration the clinical-radiologic context along with the known performance characteristics of the antibody.

The expression pattern is nuclear, which is typically moderate to strong and diffuse. The following tables show data from multiple papers in the pathology literature.

 

Liu, et al (Biocare Medical, Concord, CA)

Tumor

GATA3

GCDFP15

MGB

Breast Carcinoma 94%35-55%65-70%
ER-negativebreast ca.69% 15%35%
Urothelial Carcinoma86%


Miettinen, et al (clone L50-823, dilution of 1:500; Biocare Medical, Concord, CA)

Tumor Type

%

N

Adrenocortical Carcinoma 11%N=27
Basal Cell Ca., Skin98%N=62
Benign Skin Adnexal Tumors100%N=24
Breast, Ductal Ca., Primary92%N=179
Breast, Ductal Ca., Metastatic96%N=51
Breast, Lobular Carcinoma100%N=38
Malignant Mesothelioma58%N=64
Germ Cell Tumor, Choriocarcinoma100%N=11
Germ Cell Tumor, Endodermal Sinus Tumor100%N=6
Pancreas, Adenocarcinoma37%N=62
Renal Cell Carcinoma, Chromophobe51%N=35
Renal Oncocytoma17%N=35
Salivary Gland, Adenoid Cystic Carcinoma29%N=17
Salivary Gland, Ductal Carcinoma43%N=14
SCC - Skin81%N=31
SCC - Cervix33%N=21
SCC - Larynx16%N=36
SCC - Lung12%N=74
Urothelial Carcinoma - Low Grade100%N=22
Urothelial Carcinoma - High Grade84%N=32

No GATA-3 expression found in the following:  Seminoma (n=76), Pure Embryonal Carcinoma (n=5), Lung Small Cell Carcinoma (n=30), Lung Carcinoid (n=11), Small Intestine Carcinoid (n=18), Merkel Cell Carcinoma (n=4), Ovary Non-Serous Carcinomas (n=25), Pancreatic Neuroendocrine Tumor, Rectal Adenocarcinoma (n=27), and Thymoma (n=41). (Miettinen, et al)

GATA-3 expression in 0-5% of tumors was found in the following: Stomach Adenocarcinoma (n=133), Thyroid Papillary and Follicular Carcinomas (n=75), Renal Cell Carcinoma Not Chromophobe (n=154), Prostate Adenocarcinoma (n=95), Hepatocellular Carcinoma (n=47), and Colon Adenocarcinoma (n=142).  (Miettinen, et al)

GATA-3 expression in 6-10% of tumors was found in the following:  Anaplastic Thyroid Carcinoma (n=11), Ovarian Serous Carcinoma (n=73), Lung Adenocarcinoma (n=71), Cholangiocarcinoma (n=57), and Endometrial Adenocarcinoma (n=89).  (Miettinen, et al)

Liu, et al. (GATA-3 [HG3-31]:sc-268; Santa Cruz Biotech, Santa Cruz, CA)

Tumor

%

N

Seminoma 0%N=30
Embryonal Carcinoma0%N=24
Yolk Sac Tumor0%N=12
Lung Neuroendocrine Carcinoma0%N=61
Lung Adenocarcinoma0%N=61
Lung SCC0%N=49
Papillary Thyroid Carcinoma0%N=47
Follicular Thyroid Carcinoma0%N=37
Medullary Thyroid Carcinoma0%N=10
Clear Cell RCC0%N=82
Papillary RCC0%N=20
Colon Adenocarcinoma0%N=43
Esophageal Adenocarcinoma0%N=30
Gastric Adenocarcinoma0%N=21
Pancreatic Adenocarcinoma0%N=50
Urothelial Carcinoma86%N=72
Prostatic Adenocarcinoma0%N=136
Cholangiocarcinoma0%N=11
Breast Ductal Carcinoma91%N=99
Breast Lobular Carcinoma100%N=48
Endocervical Adenocarcinoma0%N=17
Endometrial Carcinoma2%N=96
Ovarian Serous Carcinoma0%N=56
Hepatocellular Carcinoma0%N=18
Pancreatic Endocrine Neoplasm0%N=15
Skin Melanoma0%N=100

Clark,et al

Tumor

%

N

Breast
- HR+/Her2=99%N=131
- HR+/Her2+100%N=18
- HR=/Her2+100%N=7
- Triple Negative73%N=30
Endocervix18%N=34
Vulva/Cervix SCC60%N=10
Endometrium7%N=55
Ovary10%N=50
Bladder95%N=22
Liver (cholangiocarcinoma)3%N=62
Pancreas10%N=30
Stomach2%N=62

 

GATA-3 – Breast

GATA-3 – Breast

 

References

Liu, H., Shi, J., Wilkerson, M. L., & Lin, F. (2012). Immunohistochemical evaluation of GATA3 expression in tumors and normal tissues: a useful immunomarker for breast and urothelial carcinomas. American Journal of Clinical Pathology, 138(1), 57–64. doi:10.1309/AJCP5UAFMSA9ZQBZ

 

Miettinen, M., McCue, P. A., Sarlomo-Rikala, M., Rys, J., Czapiewski, P., Wazny, K., et al. (2014). GATA3: A Multispecific But Potentially Useful Marker in Surgical Pathology: A Systematic Analysis of 2500 Epithelial and Nonepithelial Tumors. The American Journal of Surgical Pathology, 38(1), 13–22. doi:10.1097/PAS.0b013e3182a0218f

Ellis, C. L., Chang, A. G., Cimino-Mathews, A., Argani, P., Youssef, R. F., Kapur, P., et al. (2013). GATA-3 immunohistochemistry in the differential diagnosis of adenocarcinoma of the urinary bladder. The American Journal of Surgical Pathology, 37(11), 1756–1760. doi:10.1097/PAS.0b013e31829cdba7

Zhao, L., Antic, T., Witten, D., Paner, G. P., Taxy, J. B., Husain, A., et al. (2013). Is GATA3 expression maintained in regional metastases?: a study of paired primary and metastatic urothelial carcinomas. The American Journal of Surgical Pathology, 37(12), 1876–1881. doi:10.1097/PAS.0b013e31829e2525

Clark, B. Z., Beriwal, S., Dabbs, D. J., & Bhargava, R. (2014). Semiquantitative GATA-3 Immunoreactivity in Breast, Bladder, Gynecologic Tract, and Other Cytokeratin 7-Positive Carcinomas. American Journal of Clinical Pathology, 142(1), 64–71. doi:10.1309/AJCP8H2VBDSCIOBF

McCleskey BC, Penedo TL, Zhang K, Hameed O, Siegal GP, Wei S. GATA3 Expression in Advanced Breast Cancer: Prognostic Value and Organ-Specific Relapse. Am J Clin Pathol. 2015;144: 756–763. doi:10.1309/AJCP5MMR1FJVVTPK

 

E-Cadherin

 

General

E-cadherin is an intercellular adhesion marker with a membranous expression pattern. Its is commonly used is to help differentiate between DCIS (positive) and LCIS (negative). It is less helpful in differentiating invasive ductal from invasive lobular, because invasive ductal carcinoma will not infrequently lose E-cadherin expression.

If E-cadherin is positive in an invasive breast carcinoma, that would support ductal origin, but negative staining is not necessarily helpful to differentiate between a ductal or lobular process. Practically, if one is having difficulty differentiating an invasive lobular vs. invasive ductal, it is possible that E-cadherin would be negative as the cells become dis-cohesive and lose the intercellular adhesion complexes.

Up to 15% of lobular lesions may have a beaded membranous expression pattern (not continuous membrane expression seen in ductal lesions). In such equivocal cases, p120 catenin (membranous expression in ductal lesions and cytoplasmic localization in lobular lesions) or Beta-catenin may be helpful adjunct stains to help resolve discordance between morphology and E-cadherin expression. As is the case with immunohistochemistry in general, a well devised panel of antibodies for a given differential diagnosis is often the best practice, when results or the diagnosis is not straight forward.

 

DCIS vs. LCIS

IHC Stain

DCIS

LCIS

E-Cadherin +=
34BetaE12=+

Brattauer, 2002. S. Schnitt, UAMS Lecture, 2002.

 

Hematopathology

E-cadherin is a sensitive and specific marker for immature erythroid cells. It may be useful (similar to CD71) in identifying immature erythroid precursors, which may be difficult to differentiate from myeloblasts in certain circumstances. CD34 and CD117 (blast markers) may be combined with CD71 and E-cadherin (erythroid markers) to achieve this goal.

Other

E-cadherin expression has also been studied in numerous other organ tissues individually and as part of panels. Use of this stain in close conjunction with current medical literature is recommended.

 

E-Cadherin expression in a chromophobe RCC.
E-Cadherin expression in a chromophobe RCC.

E-Cadherin expression in bone marrow erythroid precursors.
E-Cadherin expression in bone marrow erythroid precursors.

E-Cadherin expression in bone marrow erythroid precursors.
E-Cadherin expression in bone marrow erythroid precursors.

E-Cadherin expression in an invasive ductal carcinoma of the breast.
E-Cadherin expression in an invasive ductal carcinoma of the breast.

E-Cadherin expression in breast DCIS.
E-Cadherin expression in breast DCIS.

E-Cadherin expression in an invasive ductal carcinoma of breast origin.
E-Cadherin expression in an invasive ductal carcinoma of breast origin.

 

References

http://www.pathologyoutlines.com/topic/stainsecadherin.html

Modern Pathology 24, 375-383 (March 2011)

Dabbs, D. J., Schnitt, S. J., Geyer, F. C., Weigelt, B., Baehner, F. L., Decker, T., et al. (2013). Lobular neoplasia of the breast revisited with emphasis on the role of E-cadherin immunohistochemistry. The American Journal of Surgical Pathology, 37(7), e1–11. doi:10.1097/PAS.0b013e3182918a2b

Klein, G. E-Cadherin Is Functionally Involved in the Maturationof the Erythroid Lineage. The Journal of Cell Biology, October 1, 1995. pp. 1–7.

Ohgami, R. S., Chisholm, K. M., Ma, L., & Arber, D. A. (2014). E-Cadherin Is a Specific Marker for Erythroid Differentiation and Has Utility, in Combination With CD117 and CD34, for Enumerating Myeloblasts in Hematopoietic Neoplasms. American Journal of Clinical Pathology, 141(5), 656–664. doi:10.1309/AJCP8M4QQTAZPGRP

Fischer S, Asa SL. Application of immunohistochemistry to thyroid neoplasms. Arch Pathol Lab Med. 2008;132: 359–372.

 

Smooth Muscle Myosin Heavy Chain

Smooth Muscle Myosin Heavy Chain (SMM-HC) is an antibody to the cytoplasmic structural protein, which is a component of the contractile apparatus in smooth muscle cells. SMM-HC is expressed in myoepithelial cells of the breast, which makes it a very useful stain in the differentiation of in situ and invasive lesions. 

One of the big problems in the breast with smooth muscle markers is that myofibroblasts may have background expression, which can occasionally mimic myoepithelial cells. SMM-HC is one of the best markers (comparatively speaking) not to have a lot background expression in myofibroblasts. Calponin and smooth muscle actin tend to have higher rates of background staining of myofibroblasts compared to SMM-HC.

SMM-HC is a more sensitive and specific marker in the differentiation of in situ and invasive breast carcinoma compared to CD10 [J Clin Pathol. 2007 Aug;60(8):958-9]. Another study noted that SMM-HC stained myofibroblasts in ~8% of cases compared to ~76% of cases for calponin. p63 is also a useful marker for breast myoepithelial cells, but the staining may not be as continuous as SMM-HC and occasional tumor cells may be positive for p63 (although the latter is recognizably different) [Am J Surg Pathol 2003 Jan;27(1):82-90]. SMM-HC combined with p63 as a dual stain may have synergistic and complementary effects.

Usefulness may be decreased in cases of DCIS, in which myoepithelial cells have been reported negative in up to 16% of DCIS cases (especially high grade DCIS). (Dewar, et al)

SMM-HC is also expressed in vascular smooth muscle cells (useful as an internal control).

 

SMM-HC – High Grade DCIS. Intact myoepithelial cell layer highlighted by smooth muscle myosin-heavy chain.
SMM-HC – High Grade DCIS. Intact myoepithelial cell layer highlighted by smooth muscle myosin-heavy chain.

SMM-HC demonstrating loss of the myoepithelial cell layer in the invasive component.
SMM-HC demonstrating loss of the myoepithelial cell layer in the invasive component.

SMM-HC highlighting the myoepithelial layer in benign breast epithelium.
SMM-HC highlighting the myoepithelial layer in benign breast epithelium.

SMM-HC highlighting vascular smooth muscle and benign duct structures.
SMM-HC highlighting vascular smooth muscle and benign duct structures.

SMM-HC highlighting expanded duct structures.
SMM-HC highlighting expanded duct structures.

 

References

Kalof AN, et. al. "Immunostaining patterns of myoepithelial cells in breast lesions: a comparison of CD10 and smooth muscle myosin heavy chain." J Clin Pathol. 2004 vol. 57(6) pp. 625-629. doi:10.1136/jcp.2003.013227

Werling RW, et. al. "Immunohistochemical distinction of invasive from noninvasive breast lesions: a comparative study of p63 versus calponin and smooth muscle myosin heavy chain." Am J Surg Pathol 2003 Jan;27(1):82-90.

Dewar, R., Fadare, O., Gilmore, H., & Gown, A. M. (2011). Best practices in diagnostic immunohistochemistry: myoepithelial markers in breast pathology. Archives of pathology & laboratory medicine, 135(4), 422–429. doi:10.1043/2010-0336-CP.1

 

GCDFP-15

 

General

Gross Cystic Disease Fluid Protein 15 (GCDFP-15) is a marker derived from breast epithelium secretions. It is a marker of apocrine differentiation. In the usual diagnostic pathology setting, GCDFP-15 is specific for breast carcinoma. Most basal-like breast carcinomas do not express GCDFP-15 or Mammaglobin. It also marks salivary and sweat gland tumors, but this is not commonly in the differential diagnosis with breast carcinoma.

The sensitivity for breast carcinomas is in the range of 50-70%. More recent data and trends have been to utilize GATA-3 more or in combination with GCDFP-15 or Mamaglobin and ER. GATA-3 has more sensitivity

Common expression patterns in carcinoma [Dennis, et al and Striebel et al]:

Tumor

(%)

Breast 50-70%
Colon<5%
Lungup to 5%
Ovary<5%
Pancreas<5%
Stomach<5%
Prostate~10%

 

GCDFP-15 – Breast Carcinoma
1. GCDFP-15 – Breast Carcinoma

Second GCDFP-15 – Breast Carcinoma
2. GCDFP-15 – Breast Carcinoma

Image of GCDFP-15 – Breast Carcinoma
3. GCDFP-15 – Breast Carcinoma

Refernece to GCDFP-15 – Breast Carcinoma
4. GCDFP-15 – Breast Carcinoma

 

References

Dennis, J. L., Hvidsten, T. R., Wit, E. C., Komorowski, J., Bell, A. K., Downie, I., et al. (2005). Markers of adenocarcinoma characteristic of the site of origin: development of a diagnostic algorithm. Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, 11(10), 3766–3772. doi:10.1158/1078-0432.CCR-04-2236

Striebel, J. M., & Yousem, S. A. (2008). Gross Cystic Disease Fluid Protein - (GCDFP-15): Expression in Primary Lung Adenocarcinoma. The American Journal of Surgical Pathology, 00(00), 7.

Lewis, G. H., Subhawong, A. P., Nassar, H., Vang, R., Illei, P. B., Park, B. H., & Argani, P. (2011). Relationship between molecular subtype of invasive breast carcinoma and expression of gross cystic disease fluid protein 15 and mammaglobin. American Journal of Clinical Pathology, 135(4), 587–591. doi:10.1309/AJCPMFR6OA8ICHNH

 

Estrogen Receptor (general information, not clone specific data)

 

Summary

  • Nuclear Marker
  • Stain is reported as PERCENT STAINING OF TUMOR CELLS and STAIN INTENSITY (1+, 2+, 3+)
  • 1% or greater nuclear expression in tumor cells is considered positive, and therefore eligible to receive hormonal therapy (CAP-ASCO guidelines).
  • CAP-ASCO recommendations are for less than 1 hr. from time of excision/biopsy to having a cut edge of tumor in 10% neutral bufferedormalin fixative. Fixation window of 6-72 hrs. These times should be noted in the pathology report (time of excision, time in gross room, and time in fixative).
  • Negative staining results in biopsy material without an internal control should be repeated on the excisional specimen using blocks with both tumor and benign breast parenchyma.

General

Estrogen Receptor (ER) is a nuclear marker, which is most commonly used to identify breast carcinomas that may be responsive hormonal therapy (e.g. Tamoxifen). It also conveys prognostic information (ER expressing tumors have a more favorable prognosis than non-expressing tumor). In normal breast duct epithelium ER will show variable patchy expression, but does not normally stain myoepithelial cells.

Interpretation

Interpretation of ER expression in breast carcinoma is based on percentage positive (+), and stain intensity (1+, 2+, 3+). Benign breast duct epithelium in the background serves as an internal barometer for stain intensity. Breast tumors are considered positive if 1% or more of tumor cells express ER (CAP-ASCO guidelines). If a case is negative for ER expression, and there is NO INTERNAL CONTROL (i.e. benign duct epithelium), which is positive, then repeat testing on the excision specimen is recommended. Block selection for testing should include benign background tissue, which can serve as an internal control.

In a breast case, if PR is positive and ER is negative, then the ER assay may not be working or the slides have been switched. PR expression should, practically, only occur in the setting of ER expression. With the implementation of proficiency testing for ER/PR/Her-2 there is a high rate of inter-institutional concordance.

Marker Specificity

Sometimes ER is utilized as a marker to resolve the primary origin of an adenocarinoma. ER is somewhat specific for breast origin, but not perfect. Any "hormonally driven organ" (i.e. ovary, breast, uterus) may commonly show ER expression. Practically any organ may show ER expression, and is best used as part of a panel for determination of primary origin of a tumor.

ER is often used as part of a panel to differentiate an endocervical adenocarcinoma (ER negative) from an endometrial adenocarcinoma (ER positive). When using ER in the evaluation of a carcinoma of unknown primary site, it should be interpreted as part of a larger panel due to its limited specificity. 

Common expression patterns in carcinoma (Dennis, JL, et al)

Tumor

Expression (%)

Breast 30-60%
Colon<5%
Lung<10%
Ovary10-50%
Pancreas0%
Stomach<5%
Prostate~10%

 

ER expression in an invasive ductal carcinoma of the breast.
ER expression in an invasive ductal carcinoma of the breast.

ER expression in an invasive breast cancer.
ER expression in an invasive breast cancer.

ER expression in benign ductal structures of the breast.
1. ER expression in benign ductal structures of the breast.

2.0 ER expression in benign ductal structures of the breast.
2. ER expression in benign ductal structures of the breast.

 

References:

Yaziji, H., Taylor, C. R., Goldstein, N. S., Dabbs, D. J., Hammond, E. H., Hewlett, B., et al. (2008). Consensus recommendations on estrogen receptor testing in breast cancer by immunohistochemistry. Applied Immunohistochemistry & Molecular Morphology : AIMM / Official Publication of the Society for Applied Immunohistochemistry, 16(6), 513–520. doi:10.1097/PAI.0b013e31818a9d3a

Hammond, M. E. H., Hayes, D. F., Dowsett, M., Allred, D. C., Hagerty, K. L., Badve, S., et al. (2010). American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Archives of Pathology & Laboratory Medicine, 134(7), e48–72.

Dennis, J. L., Hvidsten, T. R., Wit, E. C., Komorowski, J., Bell, A. K., Downie, I., et al. (2005). Markers of adenocarcinoma characteristic of the site of origin: development of a diagnostic algorithm. Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, 11(10), 3766–3772. doi:10.1158/1078-0432.CCR-04-2236

Jorns JM, Healy P, Zhao L. Review of estrogen receptor, progesterone receptor, and HER-2/neu immunohistochemistry impacts on treatment for a small subset of breast cancer patients transferring care to another institution. Arch Pathol Lab Med. 2013;137: 1660–1663. doi:10.5858/arpa.2012-0670-OA

Apple S, Pucci R, Lowe AC, Shintaku I, Shapourifar-Tehrani S, Moatamed N. The effect of delay in fixation, different fixatives, and duration of fixation in estrogen and progesterone receptor results in breast carcinoma. Am J Clin Pathol. 2011;135: 592–598. doi:10.1309/AJCPB1RIT5YXMRIS

 

Progesterone Receptor (general information, not clone specific)

Progesterone receptor (PR) is used along with estrogen receptor (ER) as a prognostication marker in breast carcinoma. Expression is determined by percent positive tumor cells and intensity (1+, 2+, 3+). PR is not as important of a prognostic marker compared to ER, but lower grade more differentiated tumors tend to show stronger expression of both ER and PR, and also have a better relative treatment outcome compared higher grade tumors.

A PR positive, ER negative, expression pattern should raise concern that either the slides were mislabeled, or the ER assay may not be performing properly. Most believe that ER expression is required for PR expression.

Outside of being used as a breast predictive marker, PR is not used significantly in routine diagnostic surgical pathology.

Summary

  • Nuclear Marker
  • Stain is reported as PERCENT STAINING OF TUMOR CELLS and STAIN INTENSITY (1+, 2+, 3+)
  • 1% or greater nuclear expression in tumor cells is considered positive (CAP-ASCP guidelines)
  • PR expression is a prognostic marker, and not directly used for eligibility to receive a specific treatment
  • PR expression without ER expression should raise significant concern that the ER and PR slides have been mixed up, or there is a problem with the ER assay. Many scientists believe that ER expression is required for PR expression.

 

PR – Metastatic Breast Carcinoma (focal expression)
PR – Metastatic Breast Carcinoma (focal expression)

PR – Breast Carcinoma
PR – Breast Carcinoma

PR – Normal Breast
PR – Normal Breast

 

References:

Hammond ME, et. al. "ASCO-CAP Guideline Recommendations for IHC Testing of ER and PR in Breast Cancer". Arch Pathol Lab Med-Vol. 134, June 2010.

Fitzgibbons PL, Murphy DA, Hammond MEH, Allred DC, Valenstein PN. Recommendations for validating estrogen and progesterone receptor immunohistochemistry assays. Arch Pathol Lab Med. 2010;134: 930–935.

Qiu J, Kulkarni S, Chandrasekhar R, Rees M, Hyde K, Wilding G, et al. Effect of delayed formalin fixation on estrogen and progesterone receptors in breast cancer: a study of three different clones. Am J Clin Pathol. 2010;134: 813–819. doi:10.1309/AJCPVCX83JWMSBNO

 

HER2 (general information, not clone specific)

HER2 (Human Epidermal growth Factor Receptor 2), which is also known as c-erb2, net, ERBB2, and CD340 is a member of the human epidermal growth factor receptor family (HER1, HER2, HER3, and HER4). Interestingly, HER1 is also known as EGFR (epidermal growth factor receptor).

HER2 is clinically significant, in that a subset of breast carcinomas (<20%) show over expression of HER2 protein. HER2 positive tumors may respond to Herceptin® (Trastuzumab), which is a monoclonal antibody inhibitor (especially effective in the neoadjuvant treatment setting). There are two methods to detect HER2 over expression: immunohistochemistry (IHC) and in situ hybridization (FISH or CISH). ISH and IHC are generally considered equivalent, but cases not positive by IHC, which do not meet fixation criteria or have 2+ (equivocal) expression, should be submitted for additional FISH or CISH analysis.

HER2 IHC Interpretation

HER2 IHC expression is membranous, and is classified as negative (0 or 1+), equivocal (2+), or positive (3+). The following are the interpretive criteria based on the 2013 CAP/ASCO guidelines:

Expression

Comment

0 (negative) No staining or faint barely perceptible staining in <10% of invasive tumor cells.
1+ (negative)Faint / incomplete barely perceptible membrane staining in >10% of invasive tumor cells.
2+ (equivocal)circumferential membrane staining weak / moderate and/or incomplete in >10% of invasive tumors cells. Strong complete membraneous staining in <10% of invasive tumor cells.
3+ (positive)Strong complete membrane staining in >10% of the invasive tumor cells.

 

Important Points

CAP-ASCO recommendations are for <1 hr. from time of excision/biopsy to having a cut edge of tumor in 10% neutral buffered fomalin fixative (necrosis time). Fixation window is now 6-72 hrs (time extended from 48 to 72 hours in the updated 2013 guidelines). Over-fixation may not be a clinically significant issue practically, but given the absence of relevant IHC data and the highly regulated environment surrounding HER2 testing, f/u FISH testing for negative results (outside the fixative window) is necessary.

Equivocal (2+) IHC results should be followed-up with an alternative (FISH/CISH) testing method, if IHC is used as the initial testing modality (most common). Less than 1/3rd of equivocal cases show Her2 over-expression by FISH analysis.

Other

HER2 testing has also been utilized in carcinomas from other primary sites as a potential therapeutic target. This is an evolving area diagnostically, and correlation with the current medical literature is important.

 

HER-2 – Score = 0
HER-2 – Score = 0

HER-2 – Score = 1+ (negative)
HER-2 – Score = 1+ (negative)

HER-2 – Score = 3+
HER-2 – Score = 3+

 

References:

Wolff, A. C., Hammond, M. E. H., Hicks, D. G., Dowsett, M., McShane, L. M., Allison, K. H., et al. (2013). Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Update. Archives of pathology & laboratory medicine. doi:10.5858/arpa.2013-0953-SA

Tafe, L. J., Janjigian, Y. Y., Zaidinski, M., Hedvat, C. V., Hameed, M. R., Tang, L. H., et al. (2011). Human epidermal growth factor receptor 2 testing in gastroesophageal cancer: correlation between immunohistochemistry and fluorescence in situ hybridization. Archives of pathology & laboratory medicine, 135(11), 1460–1465. doi:10.5858/arpa.2010-0541-OA

Ibarra JA, Rogers LW. Fixation time does not affect expression of HER2/neu: a pilot study. Am J Clin Pathol. 2010;134: 594–596. doi:10.1309/AJCPAIJPSN4A9MJI

Tafe LJ, Janjigian YY, Zaidinski M, Hedvat CV, Hameed MR, Tang LH, et al. Human epidermal growth factor receptor 2 testing in gastroesophageal cancer: correlation between immunohistochemistry and fluorescence in situ hybridization. Arch Pathol Lab Med. 2011;135: 1460–1465. doi:10.5858/arpa.2010-0541-OA

Arch Pathol Lab Med. 2001;125:746.