At times, the pathology report can be a source of frustration for both the submitting clinician and the pathologist. The goal of this lecture is to better understand the roles and responsibilities of both surgeon and pathologist, discuss times when a call to the pathologist is needed and when the use of immunohistochemistry/special stains is warranted. Just as we as clinicians would be frustrated (for lack of a better word) with a client who simply claims their pet is “sick” with no other additional information, pathologists have a similar visceral reaction to histopathology submissions forms that simply state “mass”.

1.  Surgeon responsibilities:

a.  Signalment (age, breed, sex)

b.  History including duration, prior diagnosis or therapy, etc.

c.  Physical examination, bloodwork, imaging findings

d.  Mass: gross appearance, size, location/source, duration of growth, invasiveness, etc.

e.  Type of biopsy (incisional or excisional)

f.  Ink margins and label which margins are colored, etc.

g.  Clinical impression of the case

h.  Submit all tissue removed en bloc

i.  Label lids and container and ensure proper amount of formalin

2.  Pathologist responsibilities

a.  Margin information (if excisional)

b.  ”Narrow”, incomplete, clean

c.  Should be quantified (mm or cm)

d.  Histologic grade

e.  MCT I, II, III or low grade/high grade

f.  Sarcomas, carcinoma

g.  Vascular/lymphatic invasion

h.  Mitotic index (soft tissue sarcoma, MCT, melanoma)

i.  Final diagnosis

j.  Should contain negative findings

k.  Meaning “no vascular invasion noted”

3.  There are times when a call to the pathologist is warranted as more information is needed:

a.  When the histopathologic diagnosis does not fit the clinical presentation

b.  When crucial information missing on report

c.  When all information present, but not detailed enough.

Use of Immunohistochemistry (IHC)

In veterinary oncology, the main use of IHC has been to aid the pathologist in determining the origin of the neoplastic cell population. The importance of which can’t be overstated as often poorly differentiated tumors can be very difficult to determine the exact cell of origin with routine light microscopy alone. The identification of specific antigens on the surface of cells can provide further information regarding the cell type and in some cases aggressiveness. The ever-increasing number of available antibodies is growing and is becoming commonplace in veterinary oncology.

Examples Whereby IHC is Important for Diagnosis and Therapy

1.  Amelanotic melanoma of the oral cavity (PNL2, Melan-A, TRP-1, TRP-2) vs. sarcoma

a.  Melanoma: systemic control with the Oncept® melanoma vaccine

b.  Sarcoma: systemic control with chemotherapy (doxorubicin)

2.  Synovial sarcoma (cytokeratin+, CD18-) vs. histiocytic sarcoma (cytokeratin-, CD18+)

a.  Synovial cell: if low or moderate grade local control only, if high grade doxorubicin

b.  Histiocytic sarcoma: lomustine (CCNU)

3.  Gastrointestinal stromal tumor (CD117+) vs. leiomyosarcoma

a.  If high grade; tyrosine kinase inhibitor if a GIST vs chemotherapy (doxorubicin) if a leiomyosarcoma

4.  Osteosarcoma (osteocalcin+) vs. fibrosarcoma (osteocalcin-)

a.  Fibrosarcoma: no therapy if low or moderate grade or doxorubicin if high grade

b.  Osteosarcoma: carboplatin

5.  Feline nasal carcinoma (cytokeratin+, round cell markers-) vs. lymphoma (cytokeratin+, round cell markers+)

a.  Nasal carcinoma: usually radiation alone

b.  Nasal lymphoma: combination therapy with CHOP and radiation therapy

6.  Round cell tumor

a.  Toluidine blue, chymase, tryptase (MCT)

b.  CD3 (T cell lymphoma)

c.  CD20, CD79a (B cell lymphoma)

d.  CD 18 (histiocytic sarcoma)

Tumor Markers/Panels for Prognosis

The importance of using “panels” containing several markers of malignancy is best illustrated in canine mast cell tumors. There is general consensus that the pathology and behavior of an MCT may depend substantially on the histologic pattern of the tumor, which is associated with the tumor grade. However, there is variation within the grading scheme used among pathologists which can thereby lead to subjectivity in assigning a tumor to a particular category. Traditionally, these categories are grade I (well-differentiated or low grade), grade II (intermediate grade), and grade III (poorly-differentiated or high grade). Subsequently, guiding the appropriate therapy and providing prognostic information based on the traditional grading scheme has become complicated and unpredictable. The introduction of a two-tier system (Kiupel) of low and high grade designed to minimize the subjectivity was introduced several years ago and has since been further validated in 2 additional studies.

To assist in more objectively categorizing a grade II MCT or a low grade that appears biologically high grade, various immunohistochemical and molecular tests of tumor cell proliferation are now recommended in addition to routine histopathology. These markers of proliferation are now clinically available and are readily performed on tissue biopsy samples in the form of MCT panels (www.dcpah.msu.edu/Sections/Immunohistochemistry/FAQ. php#08).

These assays provide clinicians with the ability to make more sound recommendations regarding the appropriate adjuvant therapy. This particular panel evaluates argyrophilic staining nucleolar organizing regions (AgNOR), proliferation cell nuclear antigen (PCNA), Ki-67, c-kit pattern assessment, and PCR for c-kit gene mutation. AgNOR frequency is an indirect measure of tumor cell proliferation and may be equally or even more important as the tumor grade in terms of predicting the biologic behavior of the MCT. PCNA and Ki-67 are also indirect measures of tumor cell proliferation and are most useful when interpreted in conjunction with one another.

c-kit is the protein receptor for stem cell factor found in many cells including mast cells. A genetic mutation of the c-kit gene, which encodes for the c-kit receptor itself, has been identified in some MCTs. When mutated, the receptor is constitutively active and promotes the malignant process within the cells. Mutations within the c-kit gene are associated with a more aggressive phenotype. With the new two-tier system, ideally, this should decrease the need for further panels, as low-grade tumors, for the most part, are associated with long-term survival.

If the new grading scheme may become standard of care, then the data suggest 5–15% of “low grade” tumors will behave more aggressively. Ideally, an MCT panel may help differentiate these patients and thereby guide the clinician to either a more aggressive surgery or adjuvant therapy that could benefit that patient. To run MCTs panels on all “low grade” tumors seems overkill to help identify the 5% of atypical cases, and also takes away the validity of this grading scheme in this authors opinion. Ideally, the MCT panel should be reserved for cases in which the biopsy results do not fit the clinical picture; i.e., a more aggressive site (muzzle), fast-growing tumor, severely ulcerated, large tumor, lymph node involvement, etc.

For high-grade tumors, performing a portion of the panel may be useful for treatment decision. Knowing a kit mutation status in a high-grade tumor may change the treatment choice from chemotherapy to a combination of chemotherapy and a TKI, whereby a higher response rate is noted in kit mutation-positive patients. Currently, studies are underway utilizing mutation status to help logically guide treatment decision for a more personalized medicine approach, albeit on a very basic level.

The Use of Tumor Markers for Targeted Therapies

On a basic level, this is being performed with kit mutation analysis in which results may play a role in the use of tyrosine kinase inhibitor. In the future assessment of HER-2/Neu expression in dogs with osteosarcoma could influence the decision to utilize targeted immunotherapy. Currently, the Aratana listeria based canine osteosarcoma vaccine has conditional licensure and one could see a scenario whereby its use would be limited to those tumors expressing the oncoprotein. Similarly, the use of are COX-1/COX-2 and vascular endothelial growth factor (VEGF) receptor analysis may aid not only in prognosis but the use of NSAIDs and tyrosine kinase inhibitors (Palladia™).

Understanding the responsibilities of both clinician and pathologist is needed to ensure that we, as clinicians obtain the information needed to best serve our client and patients. We are entering a new era in veterinary pathology in which panels of markers may help us better predict prognosis for a variety of neoplasias.

References

1.  Kamstock DA, Ehrhart EJ, Getzy DM, et al. Recommended Guidelines for Submission, Trimming, Margin Evaluation, and Reporting of Tumor Biopsy Specimens in Veterinary Surgical Pathology. Vet Pathol 2011;48:19–31.

2.  Smedley RC, Lamoureux J, Slefhe DG, et al. Immunohistochemical Diagnosis of Canine Oral Amelanotic Melanocytic. Vet Pathol 2011;48:32–40.

3.  Fernandez NJ, West KH, Jackson ML, et al. Immunohistochemical Stains for Differentiation Canine Cutaneous -:Round Cell Tumors. Vet Pathol. 2005;42:437–445.

4.  McCaw DL. Tumors of the Skin, Suboutis, and Other Soft Tissues: Section D – Mast Cell Tumors. In: Henry CJ, Higgenbotham ML, eds. Cancer Management in Small Animal Practice. St. Louis: Elsevier, 2010:317–321.

5.  Kiupel M, Webster JD, Bailey KL, et al. Proposal of a 2-tier Histological Grading System for Canine Cutaneous Mast Cell Tumors to More Accurately Predict Biologic Behavior. Vet Pathol. 2011;48:147–55.

6.  Sabattini S, Scarpa F, Berlato D, et al. Histologic Grading of Canine Mast Cell Tumors: Is 2 Better than 3? Vet Pathol. 2014;52:70–72.

7.  Smith J, Kiupel M, Famelly J, et al. Recurrence Rates and Clinical Outcome for Dogs with Grade II Mast Cell Tumours with a Low AgNOR Count and Ki67 Index Treated with Surgery Alone. Veterinary and Comparative Oncology. 2017;15:36–45.