1

Introduction to Neoplasia and Immunohistochemistry

Philippe Labelle, DVM, DACVPAntech Diagnostics

13th Biannual William Magrane Basic Science Course in Veterinary and Comparative Ophthalmology

2

General Considerations

• Epithelial cells

• Mesenchymal cells

– Spindle cells

– Round/leukocytic cells

NeoplasiaNeoplasia

EpithelialEpithelial SpindleSpindle RoundRound

3

Epithelial Neoplasms

• Glands• Nests• Trabeculae• Cords

• Cuboidal, columnar or polygonal• Distinct borders• Mature cells have abundant cytoplasm; Basal cells have 

less cytoplasm• Neuroepithelium often forms packets and usually has 

features similar to epithelium (not from surface ectoderm)

4

Epithelial Neoplasms

5

Epithelial Neoplasms

6

Mesenchymal‐Spindle Neoplasms

• Spindle 

• Streams

• Bundles

• Whorls

• Indistinct borders

• Scant cytoplasm

7

Mesenchymal‐Spindle Neoplasms

8

Mesenchymal‐Spindle Neoplasms

9

Round Cell/Leukocytic Neoplasms

• Round• Sheets

• “round cell” is use both for a category of cells (leukocytes) as well as a descriptor of cell shape

• Histiocytic sarcoma is a common exception. The cells may be round, spindle or polygonal/epithelioid, often in combination

• Melanocytic neoplasia can have a round cell phenotype (not of leukocytic origin)

10

Round Cell/Leukocytic Neoplasms

Lymphoma                PlasmacytomaMast cell tumor          Histiocytoma

11

Ocular Neoplasia

• The rules are similar to other organs• There are many exceptions

• Iris and ciliary epithelium (neuroectoderm) share some but not all features of epithelial (ectoderm) cells

• Lens epithelium takes on a mesenchymal phenotype

• Melanocytic neoplasia does not neatly fit in any category

12

Ocular Neoplasia

Miller PE and Dubielzig RR. Ocular Tumors. In: Withrow and MacEwen: Small Animal Clinical Oncology. p598, 2013

13

Ocular Neoplasia

Dubielzig RR. Tumors of the Eye. In: Meuten D: Tumors in Domestic Animals. 5th edition. p902, 2017.

14

Ocular Neoplasia

15

Immunohistochemistry

• Polyclonal and monoclonal

• Fixation

• Antigen retrieval

• Indirect method

• Detection

• Ramos‐Vara JA. Vet Path 2005; Ramos‐Vara JA et al. Vet Path 2014; Labelle P et al. Vet Path 2012

16

Immunohistochemistry

3,3′diaminobenzidine tetrachloride (DAB) 

3‐Amino‐9‐ethylcarbazole (AEC) 

17

Immunohistochemistry

• Read as positive vs negative 

– Not every neoplastic cell will be positive

• Read as percentage or absolute number of cells

• Distribution

• Localization

• Intensity

18

ImmunohistochemistryMNF116     Factor VIII

19

Immunohistochemistry

Lymphoma      CD3CD79a              Neg

20

Immunohistochemistry

Factor VIII

21

ImmunohistochemistryMelanoma Dx panel CD3 CD204

22

Immunohistochemistry

23

Immunohistochemistry

CD79a

24

CD3

25

Immunohistochemistry

Ki‐67

26

Immunohistochemistry

• Common antibodies• Cytokeratin• Vimentin• Melanocytic markers (melan‐A, PNL‐2, TRP1‐2) • Muscle markers (smooth muscle actin, actin, desmin) 

• Neuro/neuroendocrine markers (S100, NSE, GFAP, SYN, PGP9.5) 

• Round/leukocytic markers (CD45, CD18, CD3, CD20/CD79a/Pax5, CD204, CD117)

27

Table 2. Previously Reported Diagnostic Immunohistochemistry of Primary Ocular Neoplasms in Dogs.

P. Labelle et al. Vet Pathol 2011;49:860-869

Copyright © by American College of Veterinary Pathologists

Immunohistochemistry

28

Immunohistochemistry

• Cytokeratin

• Intermediate filament

• Normal eye: Corneal epithelium, RPE

• Neoplasms: SCC, some iridociliary neoplasms, metastatic CA

29

Immunohistochemistry

• Vimentin

• Intermediate filament

• Normal eye: almost everything

• Neoplasms: melanocytic, iridociliary, astrocytoma, meningioma, metastatic sarcomas, lymphoma, histiocytic sarcoma 

30

Immunohistochemistry

• Melanocytic markers (melan‐A, PNL‐2, TRP‐1, TRP‐2)

• Targets in melanogenesis

• Normal eye: melanocytes, corneal endothelium, neuroepithelium, tapetum, meninges

• Case report of Adenocarcinoma of the ciliary body positive for Melan‐A in a human (Sukeda A et al. BMJ case rep 2014)

• Neoplasms: Melanocytic, iridociliary?, metastatic?

31

Immunohistochemistry

• Muscle markers (Smooth muscle actin, actin, desmin)

• Intermediate (desmin) or microfilaments

• Normal eye: constrictor, dilator, ciliary muscles; choroidal stromal cells, lens epithelium, neuroepithelium, others

• Neoplasms: leiomyoma/sarcoma, iridociliary, others?

32

Immunohistochemistry

• Neuron/neuroendocrine markers (S100, NSE, GFAP, SYN, PGP9.5)

• Normal eye: Neuroepithelium, retina, uvealstromal cells, muscles, lens epithelium

• Neoplasms: Iridociliary, astrocytoma, melanocytic

33

Immunohistochemistry

• Round/leukocytic markers (CD45, CD18, CD3, CD20/CD79a/Pax5, CD204, CD117)

• Cluster of differentiation• Normal eye: Leukocytes• CD45 panleukocytic, CD18 panleukocytic• CD3 T‐cell, CD20/CD79a/Pax5 B‐cell• CD204 histiocytic (interstitial)• CD117 (mast cell and other cells including 

melanocytes)

• Neoplasms: Lymphoma, histiocytic, etc

34

Immunohistochemistry

• Melanocytic

• Vim

• +/‐ CK

• +/‐ Melan‐A/PNL‐2/TRP1‐2

• Neuro

• Iridociliary

• Vim

• +/‐ CK

• +/‐ Melan‐A/PNL‐2/TRP1‐2?

• Neuro

35

Immunohistochemistry

• Melanocytic

• Vim

• +/‐ Melan‐A/PNL‐2/TRP1‐2

• Neuro

• Lymphoma

• Vim

• CD45 +/‐ CD18

• CD3 or CD20/CD79a/pax5

36

Immunohistochemistry

• Lymphoma

• Vim

• CD18

• CD3 or CD20/CD79a/pax5

• Round cell Post‐traumatic sarcoma

• +/‐ Vim

• CD18?

• CD3 AND/orCD20/CD79a/pax5

37

Immunohistochemistry

Immunostain Melanocytic Iridociliary Lymphoma Histiocytic 

sarcoma

Metastatic 

Carcinoma

Metastatic 

Sarcoma

Schwannoma Astrocytoma Meningioma

Cytokeratin +/‐ +/‐ X +/‐

Vimentin X X X X +/‐ X X X X

Melanocytic +/‐ +/‐ +/‐ ?

Muscle +/‐ +/‐

Neuro 

markers

X X +/‐ +/‐ X X X

CD45/CD18 X X

CD3 X

CD20 X

CD204 X

38

Immunohistochemistry

39

Immunohistochemistry

40

Immunohistochemistry

CD204