Category Archives: IHC Panels

Organ Systems, Bone Marrow, WHO Classification, IHC Panels

Immunohistochemistry – MDS

The use of immunohistochemistry (IHC) in many of the MDS syndromes is limited.  Identifying an increased blast population is one of the most useful, and may indicate a more aggressive course or transformation to acute myelogenous leukemia (AML).  Helpful IHC markers may include:
 
Stain
Comment
CD34 marks immature cells including myeloblasts.  In the setting of AML, it is ~70% sensitive.  A subset of lymphoblasts may express CD34.
CD117 is a specific myeloid marker, and marks a subset of myeloblasts.  The expression is dim, and one often must look at 20-40X to clearly see expression.  Mast cells (fried egg looking cell) will have very strong expression.
CD71 marks nucleated erythroid cells.  This may be helpful in quantitating and differentiating erythroid cells from myeloid cells.  This marker may be set-up as a double stain with CD34.
In the setting of hematopoietic cells, E-Cadherin marks immature erythroid cells.  Like CD71, E-Cadherin may be useful to differentiate immature erythroid cells from immature myeloid cells.
TdT is a sensitive lymphoblast (~95%) marker.  It is not entirely specific for lymphoblasts, but other markers can help clarify diagnostic difficulties (B and T-cell markers).  
References
Hematopathology. [edited by] Jaffe, ES. 1st. ed. Elsevier, Inc. © 2011.
Organ Systems, Gynecological, Cervix, Uterus, IHC Panels

Endometrial vs. Endocervical Adenocarcinoma

Endocervical vs. Endometrial Adenocarcinoma
Endocervical and endometrial adenocarcinomas may be morphologically indistinguishable morphologically.  The question often comes up as to whether a case represents an endometrial adenocarcinoma involving the cervix, or and endocervical adenocarcinoma involving the endometrium.  The following antibodies may be helpful in such circumstances:
 
 
Endocervical
Adenocarcinoma
Endometrial
Adenocarcinoma
Negative (7-8%+)
Positive (70-93%)
Positive (65-95%)
Usually Negative
Negative (4-20%+, 38% weak)
Strong Positive (67-90%)
Strong & Diffuse Positive (90-100%)
Patchy Positive cells (~35%)
HPV
Positive (67%)
Negative
References
AJSP 2002;26:998
 
“Endocervical vs. Endometrial Adenocarcinoma:  Update on Useful Immunohistochemical Markers.”  RT Miller,The Focus ProPath Immunohistochemistry.”   April 2003.
Organ Systems, Pulmonary, IHC Panels

Mesothelioma vs. Adenocarcinoma

Immunohistochemistry
It is generally recommended to perform two mesothelioma markers and two carcinoma markers, since there is no single sensitive and specific marker for either entity.  The data below is a snapshot of several studies.
 
IHC Marker
Adenocarcinoma
Mesothelioma
8%
100%
CK5/6 (CK5)
2%
100%
0%
93%
Thrombomodulin
14%
61-77%
N-Cadherin
30%
73%
93-100%
8%
80-100%
18%
BG8
96%
7%
81-88%
0%
84%
0%
72-74%
0%
72-85%
0%
 
Obviously the main source of adenocarcinoma in this differential setting is with a primary lung adenocarcinoma and mesothelioma.  If a metastasis is likely, then the stain performance expectations for adenocarinoma may vary significantly (e.g. metastatic ovarian serous carcinoma would likely express WT-1).
References
Marchevsky AM. Application of immunohistochemistry to the diagnosis of malignant mesothelioma. Arch Pathol Lab Med. 2008;132: 397–401. 
 
Sandeck HP, Røe OD, Kjærheim K, Willén H, Larsson E. Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry. Diagnostic pathology. 2010;5: 47. doi:10.1186/1746-1596-5-47
 
Ordóñez NG. Immunohistochemical diagnosis of epithelioid mesothelioma: an update. Arch Pathol Lab Med. 2005;129: 1407–1414.
Organ Systems, Genitourinary, IHC Panels

Bladder vs. Prostate Primary

A relatively uncommon, but occasionally encountered diagnostic dilemma is differentiating between a poorly differentiated primary urothelial carcinoma and a primary prostate carcinoma at the bladder neck.  Commonly used antibodies in the past, such as PSA and PSAP, while very specific, have shown significant loss of sensitivity in less differentiated prostate carcinomas.  Newer markers (GATA-3, S-100P, p63, p501s, NKX3.1, and PSMA) have shown great promise with improved sensitivity and excellent specificity compared to traditional antibodies (CK7, CK20, PSA, and ERG).  The article cited below does an excellent job in highlighting the comparison of these antibodies, including the pros and cons.  In an article by Goldstein, the sensitivity/expression of PSA appeared closer to 50%.
Table 1.  Prostate vs. Urothelial Carcinoma in poorly differentiated lesions at the bladder trigone (Mohanty, et. al.)
 
Antibody
Prostate
Adenocarcinoma (N=20)
Urothelial
Carcinoma (N=16)
0%
100% (16)
0%
88% (14)
0%
75% (12)
0%
56% (9)
15% (3)
75% (12)
20% (4)
63% (10)
25% (5)
0%
100% (20)
0%
100% (20)
0%
100% (20)
0%
AR
100% (20)
13% (2)
ERG
35% (7)
0%
PSA expression and other markers as a function of Gleason score (Goldstein, NS)
 
Gleason Score
PSA
CK7
CK20
PAP
     6 (N=25)
100%
0%
0%
100%
     7 (N=50)
98%
0%
2%
100%
     8 (N=54)
56%
0%
4%
65%
     9 (N=58)
52%
10%
16%
74%
     10 (N=38)
47%
13%
26%
61%
Prostate specific antigen (PSA), Prostatic acid phosphatase (PAP). Reactivity was defined as >25% positive cells.
Practicality Comment
In most immunohistochemistry labs, it is not practical to have “all” of the new and “exciting” antibodies, but there are several above, which are very practical in a general pathology practice laboratory.  GATA-3 is a very good marker for both breast and urothelial carcinomas.  Some issues of specificity have arisen, which should always be considered in the context of the differential diagnosis.  P63 is a commonly used marker in lung squamous cell carcinomas.  Additionally, one may want to consider adding one of the newer prostate antibodies (e.g. p501s, NKX3.1, or PSMA), but again one should consider the specificity of the antibody in the context of the differential diagnosis.
References
Mohanty SK, Smith SC, Chang E, et al. Evaluation of contemporary prostate and urothelial lineage biomarkers in a consecutive cohort of poorly differentiated bladder neck carcinomas. Am J Clin Pathol. 2014;142(2):173–183. doi:10.1309/AJCPK1OV6IMNPFGL.
 
Goldstein NS. Immunophenotypic characterization of 225 prostate adenocarcinomas with intermediate or high Gleason scores. Am J Clin Pathol. 2002;117(3):471–477. doi:10.1309/G6PR-Y774-X738-FG2K.
Organ Systems, IHC Panels

Prostate – IHC Stains

Prostate Adenocarcinoma
IHC Expression Characteristics
Negative
Typically negative, rarely positive.
Racemase enzyme expressed by high grade PIN and Prostate adencoarcinoma (NOT a specific marker for prostate tissue).  Relatively good specificity (~90%), but may mark benign prostate lesions.  Often used in a cocktail with p63 and HMWKs (e.g. PIN4)
>95% sensitivity for prostate adenocarcinoma (highly specific)
PSAP
Earlier marker than PSA (now used occasionally as a backup) with less specificity compared to PSA
Combination stain containing a cocktail of AMACR (red), p63, and high molecular weight cytokeratins (CK5 & CK14)
34BetaE12
HMWK that is often used to highlight the basal layer of prostate glands (positivity means it is not invasive adenocarcinoma).
HMWK which has similar characteristics as 34betaE12
Nuclear marker which highlights the basal layer of benign prostate glands.
Prostatic Duct Carcinoma
 
IHC expression is the same as prostate adenocarcinoma.  Morphology is similar to breast DCIS.  Sometimes it may be confused with a primary colon adenocarcinoma (CDX-2+) or a primary urothelial carcinoma (PSA=).
References
Diagnostic Immunohistochemistry: Theranostic and Genomic Applications. [edited by] DJ Dabbs. 3rd Edition.  Elsevier, 2010.
Organ Systems, IHC Panels

Breast – Predictive Markers

One of the most important aspects of breast cancer diagnosis is the evaluation of therapeutic markers (ER, PR, and HER2).  Ki-67 is often included in the panel as a prognostic marker.  ER expression determines eligibility to receive hormonal therapy (Tamoxifin), PR expression is a prognostic marker, and HER-2 over-expression determines eligibility to receive Herceptin®.  Diagnostically, the challenge is to consistently and accurately perform and interpret these IHC markers.
 
Estrogen Receptor (ER):
  • 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 recommendations are for <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.
Progesterone Receptor (PR):
  • 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.
  • 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.
HER-2 Overexpression (HER-2):
  • Membraneous stain
  • Stain is interpreted by combining stain intensity and percentage of tumor involvement to classify as (0, 1+, 2+, or 3+).
    • 0 (negative) = No staining or cell membrane staining in <10% of tumor cells.
    • 1+ (negative) =  Faint membrane staining (partial membrane staining) in >10% of tumor cells.
    • 2+ (equivocal) =  Weak to moderate complete membrane staining in >10% of tumor cells, or strong complete staining in <10% of invasive tumor cells.
    • 3+ (positive) =  Strong complete membrane staining in >10% of tumor cells.
  • 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.  Fixation window of 6-72 hrs.  Over-fixation is probably not 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+) results should be followed-up with FISH testing, 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.
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.
 
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 
 
Arch Pathol Lab Med. 2001;125:746.
Organ Systems, IHC Panels

Breast – IHC Specific Markers

IHC stain expression pattern for various IHC antibodies in breast carcinoma.
 
IHC Stain
Comments
50-70%
30-60%
>80%
<10%
10-25%
0%
0%
0%
Mesothelin
<10%
Clin Cancer Rs 2005;11(10) May 15, 2005.
 
When evaluating for a possible breast primary, it is usually part of a larger differential diagnosis.  CK7 and CK20 is the common starting point for carcinomas of unknown primary site (CUPS), and breast has a characteristic CK7+/CK20= profile.  Unfortunately, this pattern is not uncommon, and more specific markers need to be performed.  Two specific breast markers are GCDFP-15 and ER., but their sensitivity while good is limited.  GATA-3 is a relatively newer antibody, which shows excellent sensitivity with good specificity, and should be strongly considered to be part of an antibody panel in work-up of potential breast carcinoma cases. 
  • GCDFP-15 is very specific for breast carcinoma in the setting of CUPS, but limited sensitivity.  
  • ER expression may be highly suggestive of a breast primary (especially epidemiologically), but it is not as specific.
  • Other female organs (ovary/uterus) not uncommonly express ER, and practically any tissue can occasionally excess ER.
  • GATA-3 appears to have excellent sensitivity (>90%) with good (not perfect) specificity.
Liu, et al (Biocare Medical, Concord, CA)
Tumor
GATA-3
GCDFP-15
MGB
Breast Carcinoma
94%
35-55%
65-70%
ER-negative breast ca.
69%
15%
35%
Urothelial Carcinoma
86%
 
 
 
Breast Metastasis vs. Ovarian Ca. Primary
Tumor
WT-1
CA-125
GCDFP-15
Primary Ovarian Ca.
(N=41)
76%
73%
0%
Metastatic Breast Ca.
(N-40)
3%
10%
43%
P-Value
<0.001
<0.001
<0.001
Chen, USCAP, 2004
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 
 
Clin Cancer Rs 2005;11(10) May 15, 2005.
 
Chen, USCAP, 2004
Organ Systems, IHC Panels

Breast – DCIS vs. LCIS

Lobular vs. ductal differentiation in breast lesions can on occasion be problematic.  E-cadherin is well described as a marker of ductal differentiation, but aberrant expression has be reported in 2-16% of cases of lobular neoplasia.  Some authors suggest not changing the interpretation/diagnosis based on expression of E-cadherin is the morphology is consistent with lobular carcinoma.  This author is not sure why one would do the stain to begin with if the morphologic characteristics are consistent with lobular carcinoma.  34betaE12 (CK903) has been described as a sensitive and specific in showing perinuclear dot-like expression in cases of lobular carcinoma and negativity in ductal carcinomas.  The combined panel of 34betaE12 and E-cadherin makes a nice complimentary panel to evaluate between lobular and ductal differentiation in breast carcinomas when morphology alone is not clear.
 
IHC Stain
DCIS
LCIS
E-Cadherin
+
=
34betaE12
=
+
References
Liu H. Application of immunohistochemistry in breast pathology: a review and update. Arch Pathol Lab Med. 2014;138(12):1629–1642. doi:10.5858/arpa.2014-0094-RA.
 
Bratthauer GL, Moinfar F, Stamatakos MD, et al. Combined E-cadherin and high molecular weight cytokeratin immunoprofile differentiates lobular, ductal, and hybrid mammary intraepithelial neoplasias. Hum Pathol. 2002;33(6): 620–627.
Organ Systems, IHC Panels

Breast – Stromal Invasion vs. In Situ or Benign Glands

Not infrequently in breast pathology, the differential diagnosis of micro-invasion vs. DCIS or a scerlosing lesion +/- tubular carcinoma will occur.  These type of cases can be very challenging based just on H&E morphology.  Fortunately, there are multiple IHC markers, which can be helpful in identifying the myoepithelial layer.  The main pitfall in interpretation of most of these markers, is that they will also mark myofibroblasts (on occasion) in the intervening stroma.  When this occurs, and myofibrilblasts abut the glands, the staining pattern can be misinterpreted as an intact myoepithelial layer.
 
Antibody
MEC
Myofibroblasts
++++
++
++++
++
SMA
++++
+++
p63 (nuclear)
++++
–
++++
–
+++
+
++
–
SMA = Smooth Muscle Action, SMM-HC = Smooth Muscle Myosin Heavy Chain.
 
The most commonly used stains include SMM-HC, calponin, CK5, and p63.  p63 can be combined with any of the other cytoplasmic stains as part of a double stain protocol.  SMA is the most proned of the stains to also mark fibroblasts.  S-100 is NOT recommended as a myoepithelial marker.  p63 has the least association with myofibroblast staining, but expression may be discontinuous in the myoepithelial cells layer, which can lower sensitivity when used alone.  In the opinion of many breast pathologists, SMM-HC as a single stain probably has the best combined sensitivity for the myoepithelial cells with minimal myofibroblastic staining, but many will use multiple markers in difficult cases.
 
Myofibroblast Staining
Actin > Calponin > SMM-HC
 
References
Hicks DG. Immunohistochemistry in the diagnostic evaluation of breast lesions. Appl Immunohistochem Mol Morphol. 2011;19(6):501–505. doi:10.1097/PAI.0b013e31822c8a48.
 
Liu H. Application of immunohistochemistry in breast pathology: a review and update. Arch Pathol Lab Med. 2014;138(12):1629–1642. doi:10.5858/arpa.2014-0094-RA.