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Next Generation IHC The evolution of diagnostic antibody panels
Jacob WilsonTechnical ConsultantCell Marque a MilliporeSigma Company
Discussion Topics
What is a “next generation” antibody?
What are the benefits to the lab
Comparison of first and next generation antibodiesDiscussion of the first generation antibodySupporting abstracts and dataPictures
What is a “next generation” antibody?
Improvement on an existing antibody with a technology that creates a performance advantage.
Recently developed antibodies with similar utility to more common antibodiesExample: Cytokeratin 20 CDX2PLAP SALL4
Antibodies that fill a need, gap or provide valuable information for a diagnosis
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Benefits for the lab
Patient CareImprovements in sensitivityImprovements in specificityIncrease in pathologist’s confidence More accurate diagnosis
Tech FriendlyImprovements in visualizationReduction in re-cuts
Specialties
Gastrointestinal (GI) pathology
Genitourinary (GU) pathology
Soft tissue and pediatric pathology
Dermatopathology
Pulmonary pathology
Breast and gynecological pathology
Hematopathology
GI pathology
Hepatocyte specific antigen Arginase 1
Cytokeratin 20 Cadherin 17
CD117 DOG1
CDX2 SATB2
Synaptophysin Islet-1
Hepatocyte specific antigen Glutamine Synthetase
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Hepatocyte Specific Antigen
HSA or Hep-Par1
Commonly used in liver panels and undifferentiated tumor panels
Liver vs. non-liver
Labels both benign and malignant hepatocytes
Next Generation: Arginase-1
HSA & Arginase-1
Comparison of 151 cases of hepatocellular carcinoma (HCC)
Sensitivity
Tumor Differentiation Hep-Par1 Arginase-1
Well 100.0% 100.0%
Moderately 83.0% 96.2%
Poorly 46.4% 85.7%
Overall 84.1% 96.0%
CD117 (c-Kit)
Commonly used for gastrointestinal stromal tumors (GIST) and mast cell identification
Useful for the differentiation of GIST from Kaposi’s sarcoma, smooth muscle tumors, fibromatosis, and neural tumors of the GI tract.
Next Generation: DOG1
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CD117 and DOG1
Analysis of GIST and GIST with mutations in PDGFRA
PDGFRA (Platelet derived growth factor receptor alpha)
Sensitivity
DOG1 CD117 CD34
GIST 87% 74% 59%
PDGFRA 79% 9% 27%
CDX-2
Commonly used to distinguish tumors of gastrointestinal origin, most notably colorectal carcinoma
Improvement in sensitivity and specificity when compared to CK20
Nuclear visualization
Next Generation: SATB2
Cytokeratin 20, CDX2 & SATB2
Comparison of 18 medullary colon carcinoma and 1941 additional tumors and 358 normal tissues from various sites
SensitivityCK20 CDX-2 SATB2
Medullary Colon Carcinoma 28% 67% 89%SATB2
Sensitivity SpecificityColorectal Adenocarcinoma 97% 96%
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GU pathology
Uroplakin III GATA3
PLAP SALL4
PSA NKX3.1
PSA
Commonly used for prostate and prostate carcinoma
Most useful in determining prostatic origin of carcinomas in non-prostate tissues (metastatic disease).
Next Generation: NKX3.1
NKX3.1, PSA and PSAP
Comparison of 69 cases of metastatic prostate carcinoma
Sensitivity NKX3.1 PSA PSAP
Lymph Node 59/59 (100%) 58/59 (98.3%) 59/59 (100%)Distant 9/10 (90%) 7/10 (70%) 9/10 (100%)Overall 68/69 (98.6%) 65/69 (94.2%) 68/69 (98.6%)
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Uroplakin III
Commonly used for bladder tumors
Labels urothelial carcinoma, notably transitional cell carcinoma
Aids to specify bladder vs. other tumors in the differential
Next Generation: GATA3
Uroplakin III
Comparison of 55 primary cases and 35 metastatic cases
Sensitivity
Uroplakin III Percentage
Primary 29/55 52.7%
Metastatic 23/35 65.7%
Uroplakin III
Evaluation of 71 cases, grade dependent
Sensitivity
Uroplakin III
Grade 1-2 65%
Grade 3 33%
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GATA3
Evaluation of 1,110 Carcinomas
Sensitivity
GATA3 Percentage
Urothelial Carcinoma 62/72 86.1%
2/96 endometrial carcinoma
PLAP
Placental Alkaline Phosphatase
Commonly used to differentiate tumors of germ cell lineage
Pan-germ cell tumor marker
Next Generation: SALL4
PLAP & SALL4
SensitivityPLAP SALL4
Treated Malignant Germ Cell Tumor 36 cases 36 cases Diffuse Staining 14/36 27/36Weak or Focal Staining 13/36 9/36Virtually No Staining 9/36 0/36
Comparison study with PLAP and SALL4 on 36 cases of treated malignant germ cell tumors
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Soft tissue pathology
CD34 STAT6
Smooth Muscle Actin TFE3
Synaptophysin PHOX2B
CD34
Expressed on human hematopoietic cells, vascular endothelial cells and embryonic fibroblasts.
Can be used in a variety of tumors
Useful in the diagnosis of solitary fibrous tumors
Next Generation: STAT6
STAT6
231 tumors were evaluated, 60 of which were SFT
59/60 SFT cases stained positive (98%). All other tumor types were negative for STAT6 except for 3 dedifferentiated liposarcomas and 1 deep fibrous histocytoma
Concluded that STAT6 is highly sensitive and almost perfectly specific for SFT
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STAT6
This study summarized literature on SFT markers from 1990-2015
Most important markers were STAT6, CD34, Bcl-2, and CD99. Negative markers such as S100 and EMA are also useful
SFT sensitivity
CD34 Bcl-2 STAT6Pleura 90% 94% 95%Meninges 92% 89% 91%Soft tissue 85% 85% 99%
Dermatopathology
S-100 SOX-10
EMA Adipophilin
S-100
Commonly used for melanoma and neural tumors
Distinguishes melanoma from melanocytic mimics
Used often in the detection of metastatic melanoma due to high sensitivity
Many other melanoma specific markers are negative in desmoplasticmelanoma
Next Generation: SOX-10
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S-100 & SOX-10
Comparison of 78 melanomas and 77 malignant peripheral nerve sheath tumors.
Sensitivity
S100 SOX-10Melanoma 71/78 76/78
91.03% 97.44%MPNST 23/77 38/77
29.87% 49.35%
Pulmonary pathology
P63 P40
TTF-1 Napsin-A
P63 SOX2
p63
Commonly used for breast, lung and prostate
Lung squamous cell carcinoma vs. adenocarcinoma
Labels squamous cell carcinoma of the lung
Different treatments for adenocarcinoma when compared to squamous cell carcinoma
Avastin
Next Generation: p40
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p63 & p40
Comparison of 81 squamous cell carcinomas, 237 adenocarcinomas and 152 large cell lymphomas
Sensitivityp63 p40
Lung squamous carcinoma 100% 100%Lung adenocarcinoma 31% 3%Large cell lymphoma, lung 54% 0%
Specificityp63 p40
60% 98%
Breast and gynecological pathology
E-cadherin p120 catenin
GCDFP-15/Mammaglobin GATA3
P16 Heat Shock Protein 27 and Stathmin
Mammaglobin and GCDFP-15
Commonly used in breast panels
Aides in identifying breast vs. non-breast tumors
Metastatic breast tumors or tumors metastatic to the breast
Next Generation: GATA3
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Mammaglobin & GCDFP-15
Comparison of 121 breast and 544 non-breast tumors
Sensitivity
Mammaglobin Pecentage GCDFP-15 Percentage
Breast 67/121 55.4% 28/121 23.1%
Non-breast 44/544 8.1% none reported 0.0%
GATA3
Evaluation of 1,110 Carcinomas
Sensitivity
GATA3 Percentage
Breast Carcinoma 138/147 94%
P16
A.K.A. INK4a, CDKN2, CDK4IN, MTS1
Primarily to identify cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma
Activated by human papillomavirus, which is why pathologists like it
Next Generation: HSP27 and Stathmin
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P16 and HSP27
Comparison of HSP27 and p16 specimens obtained between 2002 and 2010
HSP27 p16Normal cervical tissue 63% 29%CIN I 47% 47%CIN II 75% 67%CIN III 92% 92%Cervical SCC 100% 75%
P16 and Stathmin
A total of 193 cervical samples evaluated for Stathmin, p16, Ki67 and p57.
Stathmin p16CIN I 5/56 (9%) 40/56 (71%)CIN II 5/11 (45%) 11/11 (100%)CIN III 14/15 (93%) 15/16 (94%)
Hematopathology
CD20/CD79a PAX5
Cyclin D1 SOX-11
CD23 LEF1
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Cyclin D1
A.K.A. BCL-1
Used primarily for the diagnosis of mantle cell lymphoma.
Aids in differentiating from other small b-cell lymphomas
Next Generation: SOX-11
Cyclin D1 & SOX-11
Comparison of 30 mantle cell lymphomas, 4 Cyclin D1 negative blastoid variant mantle cell lymphomas
SensitivityCyclin D1 SOX-11
Mantle Cell Lymphoma 29/30 cases 30/30 cases
97% 100%
MCL Blastoid Variant 0/4 Cases 4/4 cases 0% 100%
Recap of the benefits of next generation antibodies
Patient CareImprovements in sensitivityImprovements in specificityIncrease in pathologist’s confidence More accurate diagnosis
Tech FriendlyImprovements in visualizationReduction in re-cuts
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References
Yan, BC et al. Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms. Am J SurgPathol 2010; 34:1147-1154.
Moll R, Wu XR, Lin JH, Sun TT. Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas. Am J Pathol 1995; 147:1383–97.
Ohtsuka Y, Kawakami S, Fujii Y, Koga F, Saito K, Ando N, Takizawa T, Kageyama Y, Kihara K. Loss of uroplakin III expression is associated with a poor prognosis in patients with urothelial carcinoma of the upper urinary tract. BJU Int. 2006 Jun;97(6):1322-6.
Liu, H, et al. Immunohistochemical evaluation of GATA3 expression in tumors and normal tissues: a useful immunomarker for breast and urothelialcarcinomas. Am J Clin Pathol 2012;138:57-64.
Bora Gurel, Tehmina Ali, Elizabeth Montgomery, Shahnaz Begum, Jessica Hicks, Michael Goggins, Charles Eberhart, Douglas Clark, Charles Bieberich, Jonathan Epstein, Angelo DeMarzo. NKX3.1 as a Marker of Prostatic Origin in Metastatic Tumors. AM J Surg Pathol. 2010 Aug; 34(8): 1097-1105.
References
Bhargava, R et al. Mammaglobin vs GCDFP-15: an immunohistologic validation survey for sensitivity and specificity. Am J Clin Pathol 2007 Jan; 127(1):103-13.
Lin F et al. Cadherin17 and SATB2 are sensitive and specific immunomarkersfor medullary carcinoma of the large intestine. Arch Pathol Lab Mad. 2014; 138:1015-26.
Zeng, W et al. Cyclin D1-negative blastoid mantle cell lymphoma identified by SOX-11 expression. Am J Surg Pathol 2012; 36:214-219.
Andeen, N et al. Metastatic Treated Malignant Germ Cell Tumors: Is SALL4 a Better Marker Than Placental Alkaline Phosphatase?. Applied Immunohistochemistry & Molecular Morphology 21/4/2015.
Bita Geramizadeh, Mahsa Marzban, Andrew Churg. Role of Immunohistochemistry in the Diagnosis of Solitary Fibrous Tumor, a Review. Iran J Pathol. 2016 Summer; 11(3): 195-203.
Doyle LA, Vivero M, Fletcher CD, Mertens F, Hornick JL. Nuclear expression of STAT6 distinguishes solitary fibrous tumors from histologic mimics. Mod Pathol. 2014 Mar;27(3):390-5.
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