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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.