B72.3 (monoclonal antibody) is also known as TAG-72, as it reacts with the so-named tumor associated protein. It is expressed on many carcinoma cells, and is sometimes part of a panel to differentiate lung adenocarcinoma (+) from mesothelioma (=). It has also been used in cytology effusion specimens. B72.3 stains in a membraneous and cytoplasmic pattern. B72.3 is expressed in high percentage of adenocarcinomas compared to extremely rare expression in mesotheliomas.
Ordonez, NG discusses the expression pattern of B72.3 in the context of multiple large studies with the expression characteristics highlighted in the table below. It is important to evaluate the expression pattern in the setting of the differential diagnosis. As example, renal cell carcinomas do not react with B72.3, and this antibody would not be helpful in a strategy to differentiate a mesothelioma from renal cell carcinoma.
Tumor
No.
Expression (%+)
Epitheliod Mesothelioma
57
3.5%
Mesothelioma
175
2%
Adenocarcinomas (various origins)
211
80.5%
Pulmonary Adenocarcinoma
110
81%
Ovarian Serous Carcinoma
45
87%
Renal Cell Carcinomas
0%
Reference
Riera, J.R., Astengo-Osuna, C., Longmate, J.A., Barrifora, H., The immunohistochemical diagnostic panel for epithelial mesothelioma: a reevaluation after heat-induced epitope retrieval. Am J Surg Pathol. 1997;21:1409.
Ordóñez, N. G. (2005). Immunohistochemical diagnosis of epithelioid mesothelioma: an update. Archives of Pathology & Laboratory Medicine, 129(11), 1407–1414.
BOB.1 (B-cell Oct-binding protein 1) is a nuclear marker whose expression is primarily restricted to B-cells (germinal center, mantle zone, and plasma cells). Cases of Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL) typically expressed both OCT2 and BOB.1. The vast majority of cases of Classical Hodgkin Lymphoma (CHL) will be negative for both markers, and a small minority (~20%) will express with one of the two markers. BOB.1 has also been found helpful to differentiate between Mediastinal Large B-Cell Lymphoma and CHL.
Annexin A1 is antibody to the ANXA1 gene product (cell membrane), and is normally expressed in T-cells, macrophages, and myeloid cells. It is a useful marker for Hairy Cell Leukemia. This marker is better than the TRAcP antibody, and helps to distinguish HCL from other mimics (splenic lymphoma with villous lymphocytes and marginal zone lymphoma).
It should be noted that with the identification of BRAF mutations in hairy cell leukemia and other markers (and flow cytometry) this antibody is not utilized widely.
AMACR (alpha-methylacyl coenzyme) is a racemese most commonly used in the diagnosis of prostate cancer. AMACR shows excellent (though not perfect) sensitivity and specificity for prostate adenocarcinoma in prostate tissue. There are several pitfalls to be aware of with AMACR in the non-prostate gland setting. Expression is seen in normal hepatocytes, proximal renal tubule epithelium, and bronchial epithelium. Metanephric adenoma of the bladder has also been reported to be positive for AMACR.
From a renal carcinoma standpoint, AMACR is expressed in a high percentage of papillary RCCs and translocation associated RCC (TFE3) (Shen, et al., Wilkerson, et al. & Al-Ghawi et al.), but is rarely expressed by other subtypes of RCC. Many other tumor types have been shown to express AMACR (see table below).
AMACR over expression (moderate to strong staining) in various tissue sites/types (Zhou, et. al.)
Tumor / Tissue
Expression (%)
Colorectal Adenocarcinoma
92%
Prostate Adenocarcinoma
83%
Breast Carcinoma
44%
High Grade PIN
64%
Colon Adenomas
75%
Ovarian
~60%
Melanoma
~40%
Lung
~35%
Urothelial
~30%
Renal Cell Carcinoma
~30%
Lymphoma
~30%
Carcinoma in situ of the urothelium has been shown to express AMACR in 50-78% of cases by Aron, et. al. AMACR has also been shown to have increasing expression levels in Barrett’s esophagus with dysplasia and esophageal adenocarinoma. Although it is unclear if anyone uses AMACR on a routine basis in cases of Barrett’s esophagus.
Pitfalls
An important point is to understand that AMACR is very helpful (especially as part of a panel with basal markers) in differentiating benign prostate glands from prostate adenocarcinoma, but AMACR is not a specific marker of prostate gland differentiation.
Photomicrographs
PIN-4 staining in prostate adenocarcinoma. AMACR (red) expression in neoplastic cells.
References
Shen, SS. “Role of Immunohistochemistry in Diagnosing Renal Neoplams: When Is It Really Useful?”Arch Pathol Lab Med, Vol. 136, April 2012. pp. 410-417.
Jiang, Z., Li, C., Fischer, A., Dresser, K., & Woda, B. A. (2005). Using an AMACR (P504S)/34bE12/p63 Cocktail for the Detection of Small Focal Prostate Carcinoma in Needle Biopsy Specimens. American Journal of Clinical Pathology, 123(2), 231–236. doi:10.1309/1G1NK9DBGFNB792L
Yang, X. J., Wu, C.-L., Woda, B. A., Dresser, K., Tretiakova, M., Fanger, G. R., & Jiang, Z. (2002). Expression of alpha-Methylacyl-CoA racemase (P504S) in atypical adenomatous hyperplasia of the prostate. The American Journal of Surgical Pathology, 26(7), 921–925. doi:10.1097/01.PAS.0000017328.13364.17
Aron, M., Luthringer, D. J., McKenney, J. K., Hansel, D. E., Westfall, D. E., Parakh, R., et al. (2013). Utility of a Triple Antibody Cocktail Intraurothelial Neoplasm-3 (IUN-3-CK20/CD44s/p53) and α-Methylacyl-CoA Racemase (AMACR) in the Distinction of Urothelial Carcinoma In Situ (CIS) and Reactive Urothelial Atypia. The American Journal of Surgical Pathology, 37(12), 1815–1823. doi:10.1097/PAS.0000000000000114
Al-Ghawi, H., Asojo, O. A., Truong, L. D., Ro, J. Y., Ayala, A. G., & Zhai, Q. J. (2010). Application of Immunohistochemistry to the Diagnosis of Kidney Tumors. Pathology Case Reviews, 15(1), 25–34. doi:10.1097/PCR.0b013e3181d51c70
Shi, X. Y., Bhagwandeen, B., & Leong, A. S.-Y. (2008). p16, cyclin D1, Ki-67, and AMACR as markers for dysplasia in Barrett esophagus. Applied Immunohistochemistry & Molecular Morphology : AIMM / Official Publication of the Society for Applied Immunohistochemistry, 16(5), 447–452. doi:10.1097/PAI.0b013e318168598b
Wilkerson ML, Lin F, Liu H, Cheng L. The application of immunohistochemical biomarkers in urologic surgical pathology. Arch Pathol Lab Med. 2014;138(12):1643–1665. doi:10.5858/arpa.2014-0078-RA.
ALK (Anaplastic Lymphoma Kinase) is a transmembrane molecule that is only normally expressed in some neural tissues. It has characteristic expression in a significant proportion of Anaplastic Large Cell Lymphoma (ALCL) cases. It has also been expressed in cases of pleomorphic liposarcoma, inflammatory myofibroblastic tumor, Merkel cell carcinoma, and a small subset of diffuse large B-cell lymphomas.
A subset (1-5%) of non-small cell lung carcinomas (usually adenocarcinomas) have an ELM4-ALK mutation, which is often sensitive to the tyrosine kinase inhibitor (TKI) crizotinib (Pfizer). Newer TKIs, including ceritinib (Novartis) and alectinib (Hoffmann-La Roche), have also been found to be effective.
Historically, ALK translocations have been identified by FISH analysis. IHC is also an accepted method with an FDA approved test (IHC CDx Assay). PCR is being studied as an alternative. ALK antibody clones 5A4 (Novocastra, Leica Biosystems, Buffalo Grove, Illinois), ALK1 (Dako, Santa Clara, California), 1A4 (Origene, Rockville, Maryland) and D5F3 (Cell signaling Technology, Danvers, Massachusetts) have been successfully used to identify ALK mutated lung tumors, with the 5A4 & D5F3 having equivalent sensitivity.
The ALK1 clone is not as sensitive and the 1A4 clone lacks specificity compared to other antibodies. Please consult the current medical literature for FDA approved tests for ALK translocation identification in non-small cell lung carcinomas.
ALK may stain in a cytoplasmic and/or nuclear pattern. In ALCL the combined pattern of cytoplasmic and nuclear staining is associated with the t(2;5).
In lung ALK staining/expression is cytoplasmic. Like other markers (e.g. Napsin A), staining may be present in macrophages. Necrotic tumor, extracellular mucin, and cells of neural origin may also stain.
Photo Gallery
ALK expression in anaplastic large cell lymphomaALK expression in anaplastic large cell lymphoma
References
Thunnissen E, Allen TC, Adam J, Aisner DL, Beasley MB, Borczuk AC, et al. Immunohistochemistry of Pulmonary Biomarkers: A Perspective From Members of the Pulmonary Pathology Society. Arch Pathol Lab Med. 2018;142: 408–419. doi:10.5858/arpa.2017-0106-SA
Chan, J. K. C., Ip, Y. T., & Cheuk, W. (2013). The Utility of Immunohistochemistry for Providing Genetic Information on Tumors. International Journal of Surgical Pathology, 21(5), 455–475. doi:10.1177/1066896913502529
Chan, J. K. C. (2013). Newly Available Antibodies With Practical Applications in Surgical Pathology. International Journal of Surgical Pathology, 21(6), 553–572. doi:10.1177/1066896913507601
Paik, J. H., Choe, G., Kim, H., Choe, J.-Y., Lee, H. J., Lee, C.-T., et al. (2011). Screening of Anaplastic Lymphoma Kinase Rearrangement by Immunohistochemistry in Non-small Cell Lung Cancer: Correlation with Fluorescence In Situ Hybridization. Journal of Thoracic Oncology : Official Publication of the International Association for the Study of Lung Cancer, 6(3), 466–472. doi:10.1097/JTO.0b013e31820b82e8
Yi, E. S., Boland, J. M., Maleszewski, J. J., Roden, A. C., Oliveira, A. M., Aubry, M.-C., et al. (2011). Correlation of IHC and FISH for ALK Gene Rearrangement in Non-small Cell Lung Carcinoma: IHC Score Algorithm for FISH. Journal of Thoracic Oncology : Official Publication of the International Association for the Study of Lung Cancer, 6(3), 459–465. doi:10.1097/JTO.0b013e318209edb9
Mino-Kenudson, M., Chirieac, L. R., Law, K., Hornick, J. L., Lindeman, N., Mark, E. J., et al. (2010). A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry. Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, 16(5), 1561–1571. doi:10.1158/1078-0432.CCR-09-2845
Reichard, K. K., McKenna, R. W., & Kroft, S. H. (2007). ALK-positive diffuse large B-cell lymphoma: report of four cases and review of the literature. Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc, 20(3), 310–319. doi:10.1038/modpathol.3800742
Medeiros, L. J., & Elenitoba-Johnson, K. S. J. (2007). Anaplastic Large Cell Lymphoma. American Journal of Clinical Pathology, 127(5), 707–722.
Takeuchi K, Choi YL, Togashi Y, et al. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer.Clin Cancer Res. 2009;15(9):3143–3149. doi:10.1158/ 1078-0432.CCR-08-3248.
AE1/AE3 is often referred to as “pan” cytokeratin, and is most commonly used to identify carcinomas, which present as morphologically undifferentiated malignant neoplasms. AE1/AE3 is also commonly used to identify micrometastatsis in sentinel lymph nodes, bone marrow, etc, and is probably the most used screening keratin antibody cocktail.
The AE1/AE3 cocktail contains CK1-8, 10, 14-16, and 19. It does not contain CK17 or CK18. This is why CAM5.2 may also used in a pan-CK cocktail. Given that AE1/AE3 is not completely sensitive for “all” cytokeratins, if a suspected carcinoma or undifferentiated tumor does not express AE1/AE3, then additional cytokeratin markers (e.g. CAM5.2 and/or 34BetaE12) may be helpful to maximize keratin expression sensitivity.
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.
Microscopic Images
AE1/AE3 highlighting an invasive adenocarcinoma.AE1/AE3 expression in a poorly differentiated sigmoid colon adenocarcinoma with signet ring features.AE1/AE3 expression in a thymoma.AE1/AE3 expression in a metastatic renal cell carcinoma (more variable expression).AE1/AE3 (pancytokeratin) expression in small bowel tissue.
Hadi, AIMM Annual Meeting, “The Thirty Most Important Antibodies”, presentation, 2011.
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
CD20 is a pan B-cell marker and is one of the most utilized hematopathology stains. The staining pattern morphology is membraneous and cytoplasmic. The sensitivity and specificity for mature B-cell neoplasms is very high.
CD19 is a B-cell marker. Until recently this antibody has only been available for use in flow cytometry. Now CD19 is available as an immunohistochemistry (IHC) marker, with similar sensitivity for B-cells to CD20. The IHC expression pattern is cytoplasmic and membranous. It is normally expressed on both Precursor B-cells and B-cells. CD19 combined with TdT would be specific to detect precursor B-cells. CD20 is not expressed on precursor B-cells, but mature B-cell with surface light chain expression (usually).
CD15 (Leu-M1) is a hematopoietic antigen expressed by granulocytes, monocytes, Reed-Sternberg cells, and subsets of T and B-cells. CD15 may also be expressed in glandular or neuroendocrine epithelial cells as well as part of a panel to distinguish between metastatic adenocarcinoma (positive) and mesothelioma (negative).
CD15 Expression
Neutrophils (~90%)
Monocytes (30-60%)
Promyelocytes
Adenocarcinoma (72%)
Classical Hodgkin Lymphoma cells
T-cell Lymphomas (subset, MF)
Acute Myelogenous Leukemia (particularly with monocytic differentiation)
CD15 stains strongly the cytoplasm of granulocytes. CD15 is most commonly used as a diagnostic marker for classical Hodgkin lymphoma (CHL). The combined staining of CD15 and CD30 in the appropriate morphologic setting is very specific for Hodgkin Lymphoma. The biggest issue with this marker in the diagnosis of CHL is sensitivity. In a well-optimized setting using a preferred clone, the sensitivity can be fairly high, ~70%. In general when CHL cases are positive for CD15, it will stain a subset of the CD30 positive cells.
When this marker is not well optimized and/or using a suboptimal clone, then the negative results can not be trusted (even when expressed in background granulocytes). Comparison analysis with a reference lab to compare IHC expression in validation cases is recommended for this antibody. PAX-5 is also a helpful marker, when CD15 is negative and CHL is suspected, as it will be dimly expressed (compared to background B-cells) in ~90% of CHL cases.
Bone Marrow
CD15 will stain at least a subset of normal granulocytes(~90%), monocytes (30-60%), promyelocytes in the bone marrow. A subset of cases of acute myelogenous leukemia (AML) will express CD15. CD15 is a myeloid marker, and appears to be preferentially expressed in AML cases with monocytic differentiation (not entirely sensitive or specific). Flow cytometry is more sensitive in detected CD15 expression than immunohistochemistry (IHC). A subset of neoplastic T and B-cells may also express CD15.
CD15 has been used as part of a panel to differentiate between mesothelioma and adenocarcinoma. CD15 is expressed in ~7% of epithelioid mesotheliomas and is not known to be expressed in sarcomatous mesothelioma while being expressed in a majority of adenocarcinomas (~72%). CD15 is not typically one of the main markers used in the mesothelioma vs. adenocarcinoma panel (there are other markers with better sensitivity/specificity profiles), but may be helpful in certain circumstances.
CD15 Expression (Marchevsky)
Epithelial mesothelioma – 7%
Sarcomatous mesothelioma – 0%
Adenocarcinoma – 72%
Squamous cell carcinoma – 30%
Renal cell carcinoma – 25-100%
Photomicrographs
CD15 – Classical Hodgkin Lymphoma. Note dark staining granulocytes in the background.CD15 – Classical Hodgkin Lymphoma
References
Wick, MR. “Immunohistochemical approaches to the diagnosis of undifferentiated malignant tumor.”Annals of Diagnostic Pathology12(2008):72-84.
Marchevsky, A. M. (2008). Application of immunohistochemistry to the diagnosis of malignant mesothelioma. Archives of Pathology & Laboratory Medicine, 132(3), 397–401.
Dunphy CH, Polski JM, Evans HL, Gardner LJ. Evaluation of bone marrow specimens with acute myelogenous leukemia for CD34, CD15, CD117, and myeloperoxidase. Arch Pathol Lab Med. 2001;125: 1063–1069.
CD10 (a.k.a. CALLA, common acute lymphoblastic leukemia antigen) is a useful marker for cells of germinal center cell origin and is expressed during the lymphoblastic phase of development. Therefore, this marker is diagnostically helpful in several areas in hematopathology: acute lymphoblastic leukemia (ALL), follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma.
ALL will often show expression of CD10. In fact, CD10 co-expression with TdT is characteristic of ALL (additional expression of T- or B-cell markers will help further classify).
CD10 is a marker of follicle center cell origin, which is characteristic of certain lymphomas including: follicular lymphoma, Burkitt lymphoma, and a subset of DLBCLs.
CD10 can be used as part of a prognostic panel (CD10, bcl-6, and MUM-1) in DLBCL to help separate cases into germinal center and non-germinal center subtypes. The Hans’ (classifier) algorithm method is the most popular, probably due to the simplicity of the algorithm and utilization of IHC markers already present in most laboratories.
Non-Hematopathology
CD10 is a useful marker in non-lymphoid malignancies: renal cell carcinoma and hepatocellular carcinoma. CD10 will have a “bile canaliculi” pattern in HCC. CD10 will also stain endometrial stromal sarcoma, and the “brush boarder” in GI tumors.
Pitfalls
CD10 can appear to have a lot of “non-specific” staining because of staining of dendritic stomal cells. This can cause a pattern similar to reticular fibers, and many describe this as a “reticular pattern,” but the staining does not directly correlate with reticulin staining. Caution should be exercised in using this stain in isolation given its lack of specificity (see below).
CD10 Expression in tumors often studied by CD10 IHC staining
Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103: 275–282. doi:10.1182/blood-2003-05-1545
Tan, P.-H., Cheng, L., Rioux-Leclercq, N., Merino, M. J., Netto, G., Reuter, V. E., et al. (2013). Renal tumors: diagnostic and prognostic biomarkers. (Vol. 37, pp. 1518–1531). Presented at the The American journal of surgical pathology. doi:10.1097/PAS.0b013e318299f12e
Chang, C.-C., McClintock, S., Cleveland, R. P., Trzpuc, T., Vesole, D. H., Logan, B., et al. (2004). Immunohistochemical expression patterns of germinal center and activation B-cell markers correlate with prognosis in diffuse large B-cell lymphoma. The American Journal of Surgical Pathology, 28(4), 464–470.
Tan P-H, Cheng L, Rioux-Leclercq N, Merino MJ, Netto G, Reuter VE, et al. Renal tumors: diagnostic and prognostic biomarkers. 2013. pp. 1518–1531. doi:10.1097/PAS.0b013e318299f12e
Dewar R, Fadare O, Gilmore H, Gown AM. Best practices in diagnostic immunohistochemistry: myoepithelial markers in breast pathology. Arch Pathol Lab Med. 2011;135: 422–429. doi:10.1043/2010-0336-CP.1
Alizadeh AA, Elsen MB, Davis RE, Ma C. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000.
Swerdlow SH, Campo E, Pileri SA, Harris NL, Stein H, Siebert R, et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood. 2016;127: 2375–2390. doi:10.1182/blood-2016-01-643569
