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Diagnostic pathology for the cancer patient
Diagnostic Pathology for the Cancer Patient ShelleyJoy Newman, DVM, DVSc, DACVP
It is necessary to have a cooperative relationship between the clinician and the pathologist, because information from both is critical for determining the best case management and favorable prognosis. The principle roles of the veterinary pathologist are to describe the submitted tissues, to determine the nature of the tumor, and to assess completeness of surgical margins. Recently, the Council of the American College of Veterinary Pathologists (ACVP) appointed an ad hoc committee on oncology to evaluate oncologists' needs by polling members of the Veterinary Cancer Society (VCS). The committee's vision was to review morphologic, immunologic, and molecular techniques so that veterinary pathologists could meet the current and future diagnostic and prognostic needs of oncologic veterinarians. The factors determined to be most important to the VCS members in selecting a diagnostic pathology provider included, in order of importance: (1) reliability of results; (2) short turnaround time; (3) easy access for consultation; (4) rapport with pathologist; (5) convenience of service; and (6) cost of service. This manuscript will attempt to review improved morphologic, immunologic, and molecular techniques to provide for the future diagnostic and prognostic needs of oncologic veterinarians. © 2003 Elsevier Inc. All rights reserved.
here is a strong cooperative relationship required between
T the clinician and the pathologist, because information from both is critical for determining the best case management and prognosis. ~ The principle roles of the veterinary pathologist have been determined to be description of the submitted tissues, determination of the nature of the tumor, and assessment of completeness of surgical margins. 2 Determining prognosis may be more difficult because the pathologist often is involved relatively late in the course of the disease and rarely has all the relevant information to clearly summarize the case. x,3 Thus, the clinician's role is to interpret all the data and determine the therapeutic plan accordingly. Ensuring the reliability of results is a necessary goal of any quality diagnostic service, but is influenced by numerous variables, including those involving the clinician, the laboratory, and the pathologist. Submission of unlabeled containers, or containers with multiple unidentified dermal biopsy samples and incomplete or absent patient information forms are means by which clinicians contribute to unreliable results. Additionally, improper fixation ratios (less than 1:10 in 10% buffered formalin) produce tissue artifact. 4 Thus, for large specimens, the clinician should select representative samples for submission while storing the formalin-fixed specimen at the clinic or,
From the Animal Medical Center, New York, NY. Address reprint requests to Dr. Shelley Newman, The Animal Medical Center, 510 East 62nd Street, New York NY, 10021-8314. © 2003 Elsevier Inc. All rights reserved. 1096-2867/03/1802-0017530.00/0 doi:10.1053/svms.2003.36633
following complete fixation, submit the entire specimen in a smaller container. 1,5 Bouins fixative may provide superior fixation for reproductive or ocular specimens. It may be necessary for a clinician to maintain a variety of sizes of containers as well as a large container of formalin for large specimens, such as spleens or limbs. Because extent and appropriateness of tissue sampling are also critical to an accurate diagnosis, at least 3 representative sections (rather than large focal areas of hemorrhage, inflammation, or necrosis), including some that border normal tissue, are recommended, because this assists in determining the degree of invasiveness of the tumor. 1,5,6 If excisional biopsies are submitted, then margins of interest should be marked and an interpretive summary included. 1 Methods of marking surgical margins include preferential inking (India ink or fluorescent surgical marking ink), suturing, or staplingY r If the entire sample is not submitted for pathology, representative labeled margins must be sent.S Confusion occurs with inking if margins are contaminated during processing and interpreted as surgical margins. 4 The following laboratory-related factors can confound the reliability of results: loss tof samples, freezing or leakage of samples such that inadequate fixation results in tissue artifact, incorrect trimming, improper tissue orientation, disintegration of small samples, shatter artifact in osseous or mineralized tissues, collapse of tissues, ie, eye, and mislabeling of blocks or slides during processing. 1.9 Ensuring and maintaining competency of pathologists is a difficulty, as in all professions that revolve around subjective opinion. Controversies exist most commonly in diagnosis, grade, or completeness of margins. 3 For diagnosis, this may be in part due to the variable use of older terminology, referral to human pathologists untrained in veterinary pathology, difficulty in distinguishing benign tumors from well-differentiated malignant tumors, and the complications of marked inflammation and granulation tissue response in tumor diagnosis. 1 Resectioning available tissues and performing a series of special light microscopic or immunohistochemical stains may be useful in such cases. 5 Additionally, seeking multiple opinions from pathologists at larger facilities or those with specialty expertise can minimize these concerns. 5 The advent of telemedicine may allow for transfer of images via the Internet for consultation with others at a distance from the primary laboratory. All pathologists should strive to deliver a biopsy report that contains a detailed histologic description, an assessment of completeness of excision, a final diagnosis, and a grade, if applicable. 4 As determined by the ACVP ad hoc committee's oncologist survey, the components of greatest importance within a pathology report are: l) surgical margins; 2) diagnosis; 3) grade; 4) histologic description; 5) mitotic index; and 6) pathologist's opinion of prognosis)
Clinical Techniques in Small Animal Practice, Vol 18, No 2 (May), 2003: pp 139-144
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Surgical Margins The best way for a pathologist to assess surgical margins is to use visual indicators such as ink, in a variety of colors, to allow for proper tissue orientation and interpretation. Margin assessment is still subject to interpretive differences, because it is difficult to transmit 3-dimensional margins into a 2-dimensional light microscopic plane, and to conclude that all margins are free of infiltrating cells based on evaluation of a 5-/xm thick section. 4 Sometimes the clinician is better off relying on knowledge of likely clinical behavior rather than on a literal interpretation of the surgical margins. 4 A 10% recurrence rate for human soft-tissue sarcomas with complete excision is documented, although the recurrence rate is higher in veterinary medicine. ~° Completeness of margins can be described as radical, wide, marginal, or dirty. Dirty margins occur when tumor cells extend to the surgical margin. 1 Marginal (clean but close) margins occur when there is less than 1 cm of normal tissue present around the neoplasm, s Wide margins are seen when 1 to 3 cm of normal tissue surround the tumor. ~ Radical margins are attained when the entire compartment is removed, ie, amputation. 1
Diagnosis Once a diagnosis of neoplasia---defined as a disturbance of growth characterized by excessive, uncontrolled proliferation of cells---is determined, then the origin and nature of the tumor must be evaluated. Benign tumors typically resemble the normal tissue of origin, grow slowly by expansion and compression, and fail to metastasize. Life-threatening effects may still occur when these benign tumors are located in surgically compromised locations, are predisposed to rupture and hemorrhage, and are secreting hormones that affect physiologic function.1 Cellular features of benign masses include low nuclear/ cytoplasmic ratio, nuclei of uniform size and shape, and a low mitotic rate. 1,3,8,H In contrast, most malignant tumors grow rapidly and invade the surrounding tissues. Invasion may involve infiltrative cell aggregates, invasion of vascular or lymphatic spaces, or induction of a desmoplastic response as seen by excess fibrous connective tissue reaction. 1 Features of malignant neoplasms are poor cellular differentiation, high cellularity, high nuclear/cytoplasmic ratio, variation in nuclear (anisokaryosis) and cytoplasmic size (anisocytosis), variation in nuclear shape, large nucleoli, multiple nucleoli, increased necrosis, abnormal mitotic figures, and a high mitotic rate. ~ Neoplasms originate from epithelial or mesenchymal tissue. 2 Benign mesenchymal tumors originating from connective tissue, striated or smooth muscle, endothelial cells, or meninges are designated with the suffix "oma." Those of malignant origin carry the corresponding suffix "sarcoma." Similarly, benign epithelial tumors originating from glandular tissue carry the suffix "adenoma," and those that originate from solid epithelium, "papilloma." The malignant epithelial tumors are designated "adenocarcinoma" or "carcinoma," respectively. 8 Teratomas are exceptions because they are neoplasms composed of at least two different embryonic germ layers. Two neoplastic tissues can often be seen in mixed tumors in which glandular cells and myoepithelial cells proliferate, such as in mammary or salivary tumors. Additionally, there are some tumors in which histologic features do not necessarily correlate with behavior;
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canine histiocytoma, 1 infiltrative lipoma, t2 and histologically low-grade, biologically high-grade fibrosarcoma of the canine head. 13 A recent report determined that classic histologically benign canine choroidal melanomas can exhibit unexpected malignant behavior, as can conjunctival melanomas. I<~5 Approximately 90% of human cancer cases can be diagnosed by light microscopy alone. 1,~6,~r The remaining 10% require special procedures. ~ Special light microscopic stains such as toluidine blue, periodic acid Schiff PAS, alcian blue, or Masson Trichrome can identify mast-cell granules, mncopolysaccharide, ground substance, and collagen fibrils, respectively. More recently, immunohistochemistry has been more widely used to determine tissue of origin by identifying intermediate filaments and other structural cytoplasmic proteins. This procedure works on the premise that primary antibodies recognize specific cell components and then the secondary antibody is directed against the primary antibody. This complex becomes visible because the secondary antibody is linked to peroxidase or avidin biotin peroxidase complexes that catalyze a reaction to precipitate a dye on the complex. 16,18Heterogeneous tumorcell populations can yield inconsistencies in protein expression and staining, and excessive fixation can create false results by cross- linking the antibody. 1 Thus, it is best to perform panels of antibodies to obtain accurate and useful information, but even then, staining should be used only as a guide and not as a definitive indicator of neoplastic cell type. 16 Additionally, immunohistochemistry does not distinguish between benign and malignant cells. 1 The two most utilized markers for anaplastic tumors include cytokeratin for epithelial origin and vimentin for mesenchymal origin. Only rare tumors, such as mesotheliomas and synovial cell sarcomas, can stain for both markers. xg,x° Chromagranin A ig a useful marker for pancreatic tumors of neuroendocrine origin and their metastases, xx,x2Other endocrine tumors can be characterized by a series of primary products such as thyroglobin in thyroid carcinomas, calcitonin in medullary carcinomas, and thyroid transcription factor-1 in a proportion of thyroid follicular carcinomas. 23 Melan A is more specific but less sensitive than S100 for differentiating melanoma from pigmented basal cell tumor in cats, the latter of which would also be cytokeratin positive. 24 The most useful marker to determine dendritic cell origin for histiocytic sarcoma in dogs is positivity for CD18 and negativity for CD3 and CD79a. 25 Lastly, some neoplasms may revert to expressing fetal antigens, as is seen in a proportion of renal cell carcinomas that express cyclo-oxygenase (COX). a6 The accuracy of predicting prognosis, malignancy, or response to therapeutic modalities is significantly increased by the results of special procedures, and pathologists are in the best position to recommend these tests. 4 Immunohistochemistry staining techniques for proliferation markers or altered gene products, and silver staining (Ag) for nucleolar organizer regions (NORs) is fast progressing. In malignant cells, NORs desegregate from the nucleoli and disperse throughout the nucleus to permit RNA transcription.5 The size and number noted are related to nuclear activity. 5 An increase in number and area of AgNORs is correlated with cell proliferation, a The stained complex corresponds to the binding sites of RNA polymerase 1 and associated proteins regulating transcription and post-translational modification of ribosomal RNA. 8 Automated image analysis can minimize observer bias and is less time consuming and tedious than manual counting methods. 5 AgNORs have
SHELLEY JOY NEWMAN
been determined to be a valuable prognostic marker for the majority of tumors studied. Additionally, prognostic information can result from utilizing monoclonal antibodies against epitopes of antigens expressed by proliferating cells# Examples are proliferating cell nuclear antigen (PCNA) and MIB-1 antibody directed against Ki67, which are both nonhistone proteins detected in varying amounts during several phases of the cell cycle. 5 Canine malignant lymphomas with a longer disease-free interval and survival time correlated with larger mean AgNOR area, larger maximal AgNOR area, a larger total AgNOR area, shorter distance between two AgNORs, a smaller AgNOR area to nucleus ratio, and a smaller number of AgNORs per nucleus. 2r Similarly, AgNOR counts correlated inversely with response to therapy in an additional lymphoma study. 28 Canine transmissible venereal tumor with high AgNOR counts had poor chemotherapy repsonses. 29 Metastatic canine seminomas had higher AgNOR counts compared with nonmetastatic forms. 3° Canine perianal tumors had higher AgNOR counts in the malignant variantsY AgNOR counts correlated with histologic grade and prognosis following surgery in canine mammary tumors, 32 and in one study, AgNOR index for 1-year survival was the most significant parameter influencing survival. 33 Additionally, higher AgNOR scores were seen in malignant f o r r n s , 34,35 but no difference was noted between metastatic and nonmetastatic forms. 34 In feline mammary tumors, high AgNOR count was agsociated with shorter postoperative survival times, 36 but AgNOR cluster size did not correlate to mitotic index, tumor stage, nuclear atypia, or histologic appearance. 3r Mast cell tumors had a poorer prognosis if AgNOR and PCNA counts were high and c-kit was identified. 38-4° Both canine meningioma and feline squamous cell carcinoma with high PCNA levels were associated with poor responses to radiation therapy. 41,42 In canine and feline mammary tumors, high PCNA is associated with malignant t u m o r s , "~3,44 and predicted local tumor recurrence following surgery. 45 Canine mammary tumors with high Ki67 are associated with malignancy: metastasis, low disease-free survival rate, poor prognosis, and risk of death. B3,46 Ki67 is inversely correlated with estrogen receptor alpha scores. 47 Canine and feline melanotic tumors demonstrated higher Ki67 in malignant than in benign tumors. 48,49 Lack of correlation between Ki67 and postsurgery survival suggests that this marker alone is not sufficient to determine the correct prognosis in feline mammary carcinomas, and the histologic grading system actually has a higher predictive value. 5° In canine lymphoma, Ki67 correlated with histologic grade, 5~ and with prolonged first remission duration after chemotherapy. 52 Flow cytometry can be utilized to determine ploidy (the nuclear DNA content of cells), which can aid in distinguishing malignant tumors from other lesions. Aneuploidy is most associated with neoplastic transformation, and has been documented with these tumors: canine malignant melanomas, 53 osteosarcomas, 54 transitional cell carcinomas, 55 canine m a m m a r y adenocarcinomas, 56 prostate carcinomas, 5r and a minority of feline m a m m a r y adenocarcinomas. 58 There has been little correlation between ploidy and histology, survival time, or clinical characteristics for transitional cell carcinoma. 55 Presence of chromosomal aberrations, namely trisomy of chromosome 13 in canine lymphoma, produced a significantly longer first remission length and survival time. 59
DIAGNOSTIC PATHOLOGY FOR THE CANCER PATIENT
Grade The widespread usage of grading systems is fraught with difficulty because there are a variety of schemes, some of which are unfounded, and many of which utilize subjective terms such as "poorly," "moderately," or "well differentiated." Additionally, some tumors are not amenable to grading schemes. Most grading schemes are based on mitotic rate, degree of cellular differentiation, cellular or nuclear pleomorphism, and percentage of necrosis. 1 Other important features include overall cellularity, degree of invasiveness, amount of inflammation, nucleolar size, amount of stromal reaction, and lymphoid response, s.s Current grading schemes vary and may focus on one criterion, such as mitotic index, and result in low, medium, or high-grade designations, whereas others are based on a combination of criterion. 8 Only mitotic index and amount of necrosis are considered objective. I The behavior of nonangiomatous/nonlymphomatous splenic sarcomas was found to be closely correlated with mitotic index, where those with an index greater than 9 had a shorter survival time. 6° Similarly, mitotic index in combination with lymphoid:fibrohistiocytic proportion in splenic nodules were the anatomic features most predictive of postsplenectomy mortality. 61 High mitotic index and a low lymphoid:fibrohistiocytic proportions indicated a greater prObability of higher short-term mortality. 61 Frequently, cellular morphology is the most accurate predictor of behavior of a neoplasm. 8 Individual tumors may have specific associations between prognosis and morphology, and the latter is utilized in proven morphological grading schemes, s Ultimately, the grade is important for prognosis, but also for determination of the need for adjuvant therapy. 1 Analysis of lymph-node tissue can also confirm metastatic disease, which affects overall prognosis, substantiates grade, and determines stage. Fine-needle aspiration was determined to be a sensitive and specific method of evaluating the regional lymph nodes in dogs and cats because it correlated well with lymph-node histology. 62 Carcinomas showed a higher incidence of lymphnode metastases than sarcomas. 62 Grading of tumors is becoming increasingly important because of the behavior of some tumors being shown to be dependent on grade. 1 Examples where grade or histologic features predict outcome include the following: canine and feline mastcell t u m o r , 63,64 canine and feline mammary gland adenocarcinoma, 65,66 canine soft-tissue s a r c o m a s , 6r,68 canine cutaneous and ocular melanoma, 69,r° canine splenic nonhematogenous/ nonlymphomatous sarcoma, 6° synovial cell sarcoma, 2° transitional cell carcinoma, rl multilobular osteochondrosarcoma, r2 lymphoma, r3,r4 and squamous cell carcinoma of the tongue, r5 The profession agrees that improved grading schemes are required for these important tumors: lymphomas, lymphoid leukemias, nonlymphoid leukemias, histiocytic tumors, soft-tissue sarcomas, mammary tumors, and mast-cell tumors. 3 Such advanced grading schemes might incorporate AgNOR and proliferation cell marker counts. The pathologist must stay abreast of the literature regarding current grading schemes, s The most recently published histologic grading scheme is for canine osteosarcoma, and is based on histologic criterion such as cellular pleomorphism, mitoses, amount of matrix, percentage of tumor cells, and amount of necrosis. 76 Many of the more accepted schemes have been adapted from human medicine. ~ The grading system for canine soft-tissue
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sarcomas is adapted from that in humans, and tends to rely more on grade than the cell of origin. ~,6r,68 Tumors are scored for degree of differentiation, mitotic rate, and necrosis, and an overall grade assigned following addition of the numeric scores. ~,6r,68 Grade 1 encompasses a numeric score of 3 or 4, grade 2 a score of 5 or 6, and grade 3 a score of 7, 8, or 9.11,67,68 It was found that the combination of these 3 factors was necessary to result in useful prognostic information. 6r Histologic grade was the most important prognostic factor for human soft-tissue sarcomas, 6r and the grading system was reproducible among human pathologists. 68 This is suspected to be similar in veterinary medicine. In addition to grading, staging systems are intended to determine prognosis, to aid in planning treatment, and to evaluate the success of treatment regimes, s,ls The TMN system developed by the World Health Organization classifies tumors by local (T), regional (N), and metastatic (M) characteristics, s Although TO indicates no evidence of neoplasia, T1 to T4 are associated with increasing size of the primary mass. NO means lymph nodes are not involved, but N1 to N3 is utilized for increasing nodal involvement. M0 indicates an absence of metastases, which are otherwise recorded as M1 or M2. These values should be taken into consideration along with all other data to assist with determining prognosis and treatment decisions. 8
Histologic Description An ideal report should contain a detailed microscopic description, a final diagnosis, and a grade, if applicable. 4 Often, the description will assist in determining the limits of a complete clinical evaluation, proper treatment options, and an accurate prognosis. 4
Mitotic Index As an isolated result, this is not considered important but can be an integral of accepted grading schemes. Mitotic index is the most utilized criterion to indicate degree of proliferation, and in some tumors, such as melanoma and other sarcomas, this has been determined to be the best predictor of survival and/or response to treatment, although this does not hold true for histiocytomas or transmissible venereal tumors. 8
Prognosis Many feel that pathologists should not make blanket statements about prognosis because they rarely have all the clinically relevant information to do so. Additionally, much of the prognostic data are extrapolated from humans or from a relatively small body of survival study data for individual tumors in the veterinary literature. Ultimately, we should develop classification schemes using morphologic, immunologic, and cytochemical characteristics that better predict biologic behavior and response to therapy for tumors of veterinary importance.
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committee to the ACVP membership and interested pathology community. Vet Pathol 39:525-528, 2002 3. Powers B, Dernell WS: Tumor biology and pathology. Clin Tech Sm Anim Pract 13:4-9, 1998 4. Morrison WB: Understanding the biopsy report: What else to ask for, in Morrison WB (ed): Cancer in Dogs and Cats: Medical and Surgical Management (ed 1). Baltimore, MD, Lippincott, Williams & Wilkins, 1998, pp 139-146 5. Withrow SJ: Biopsy principles, in Withrow SJ, MacEwen EG (eds): Small Animal Clinical Oncology (ed 2). Philadelphia, PA, Saunders, 1989, pp 52-57 6. Salisbury SK: Principles of the surgical biopsy of cancer, in Morrison WB (ed): Cancer in Dogs and Cats: Medical and Surgical Management (ed 1). Baltimore, MD, Lippincott Williams & Wilkins, 1998, pp 131-137 7. Rochat MC, Mann FA, Pace LW, et al: Identification of surgical biopsy borders by use of India ink. J Am Vet Med Assoc 201:873878, 1992 8. Cullen JM, Page R, Misdorp W: An overview of cancer pathogenesis, diagnosis and management, in Meuten DJ (ed): Tumors in Domestic Animals (ed 2). Ames, Iowa, Iowa State Press, 2002, pp 28-44 9. Madewell BR: Cellular proliferation in tumors: A review of methods, interpretation, and clinical applications. J Vet Intern Med 15:334-340, 2001 10. Mandard AM, Petiot JF, Marnay J, et al: Prognostic factors in soft-tissue sarcomas: A multivariate analysis of 109 cases. Cancer 63:1437-1451, 1989 11. Powers BE, Hoopes PJ, Ehrhart EJ: Tumor diagnosis, grading and staging. Sem Vet Med Surg (Sm Anim) 10:158-167, 1995 12. Bergman PJ, Withrow SJ, Straw RC, et al: Infiltrative lipoma in dogs: 16 cases (1981-1992). J Am Vet Med Assoc 205:322-324, 1994 13. Ciekot PA, Powers BE, Withrow SJ, et al: Histologically low-grade, yet biologically high-grade, fibrosarcomas of the mandible and maxilla in dogs: 25 cases (1982-1992). J Am Vet Med Assoc 204:610615, 1994 14. Hyman JA, Koch SA, Wilcock BP: Canine choroidal melanoma with metastases. Vet Ophtho 5:113-117, 2002 15. Collins BK, Collier LL, Mill,er MA, et al: Biologic behavior and histologic characteristics of canine conjunctival melanoma. Prog Vet Comp Ophtho 3:135-140, 1993 16. Pfeifer JD, Wick MR: The pathologic evaluation of neoplastic diseases, in Murphy GP, Lawrence W, Lenhard RE (eds): American Cancer Society Textbook of Clinical Oncology (ed 2). Atlanta, GA, American Cancer Society, 1995, pp 75-95 17. Pfeifer JD, Wick MR: The pathologic evaluation of neoplastic diseases, in Holleb, AL, Fink DF, Murphy GP (eds): Clinical Oncology (ed 1). Atlanta, GA, American Cancer Society, 1991, p 7 18. Hsu S-M, Raine L, Fanger H: Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase staining techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580, 1981 19. McDonough SP, MacLachlan NJ, Tobias AH: Canine pericardial mesothelioma. Vet Pathol 29:256-260, 1992 20. Vail DM, Powers BE, Getzy DM, et al: Evaluation of prognostic factors for dogs with synovial sarcoma: 36 cases (1986-1991). J Am Vet Med Assoc 205:1300-1307, 1994 21. Myers NC, Andrews GA, Chard-Bergstrom C: Chromagranin A plasma concentration and expression in pancreatic islet cell tumors of dogs and cats. Am J Vet Res 58:615-620, 1997 22. Doss JC, Grone A, Capen CC, et al: Immunohistochemical localization of chromagranin A in endocrine tissue and endocrine tumors of dogs. Vet Pathol 35:312-315, 1998 23. Ramos-Vara JA, Miller MA, Johnson GC, et al: Immunohistochemical detection of thyroid transcription factor 1, thyroglobulin and calcitonin in canine normal, hyperplastic and neoplastic thyroid gland. Vet Pathol 39:480-487, 2002 24, Ramos-Vara JA, Miller MA, Johnson GC, et al: Melan A and $100 protein immunohistochemistry in feline melanomas: 48 cases. Vet Pathol 39:127-132, 2002 25. Affolter VK, Moore PF: Localized and disseminated histiocytic sarcoma of dendritic cell origin in dogs. Vet Pathol 39:74-83, 2002 26. Khan KNM, Stanfield KM, Trajkovic D, et al: Expression of cyclooxygenase-2 in canine renal cell carcinoma. Vet Pathol 38:116-119, 2001 SHELLEY JOY NEWMAN
27. Kiupel M, Testke E, Bostock D: Prognostic factors for treated canine malignant lymphoma. Vet Pathol 36:292-300, 1999 28. Vail DM, Kisseberth WC, Obradovich JE, et al: Assessment of potential doubling time (1- pot) argyrophilic nucleolar organizer regions (AgNOR) and proliferating cell nuclear antigen (PCNA) as predictors of therapy response in canine non-Hodgkins's lymphoma. Exp Hematol 24:807-815, 1996 29. Harmelin A, Zuckerman A, Nyska A: Correlation of Ag-NOR protein measurements with prognosis in canine transmissible venereal tumor. J Comp Pathol 112:429-433, 1995 30. DeVice G, Papparella S, Di Guardo G: Number and size of silverstained nucleoli (AgNOR clusters) in canine seminomas: Correlation with histological features and tumor behavior. J Comp Pathol 110: 267-273, 1994 31. Preziosi R, Salda LD, Ricci A, et al: Quantification of nucleolar organizer regions in canine perianal gland tumors. Res Vet Sci 58:277-281, 1995 32. Bostock DE, Moriarty J, Crocker J: Correlation between histologic diagnosis, mean nucleolar organizer region count and prognosis in canine mammary tumors. Vet Pathol 29:381-385, 1992 33. Sarli G, Preziosi R, Benazzi C, et al: Prognostic value of histologic stage and proliferative activity in canine malignant mammary tumors. J Vet Diagn Invest 14:25-34, 2002 34. Bratulic M, Grabarevic Z, Artukovic B, et al: Number of nucleoli and nucleolar organizer regions per nucleus and nucleolus--Prognostic value in canine mammary tumors. Vet Pathol 33:527-532, 1996 35. Destexhe E, Vanmanshoven P, Coignoul F: Comparison of argyrophilic nucleolar organizer regions by counting and image analysis in canine mammary tumors. Am J Vet Res 56:185-187, 1995 36. Castagnaro M, Casalone C, Nervi GC, et al: Argyrophilic nucleolar organizer regions (AgNOR's) count an indicator of post-surgical prognosis in feline mammary carcinomas. Res Vet Sci 64:97-100, 1998 37. De Vice G, Maiolino P, Restucci B: Silver stained nucleolar (AgNOR) cluster size in feline mammary carcinomas: Lack of correlation with histological appearance, mitotic activity, tumor stage and degree of nuclear atypia. J Comp Pathol 113:69-73, 1995 38. Simoes JPC, Schoning P, Butine M: Prognosis of canine mast cell tumors: A comparison of three methods. Vet Pathol 31:637-647, 1994 39. London CA, Kisseberth WC, Galli SJ, et al: Expression of stem cell factor receptor (c-kit) by the malignant mast cells from spontaneous canine mast cell tumors. J Comp Path 115:399-414, 1996 40. Zemke D, Yamini B, Yuzbasiyan-Gurkan V: Mutations in the juxtamembrane domain of c-KIT are associated with higher-grade mast cell tumors in dogs. Vet Pathol 39:529-535, 2002 41. Theon AP, LeCouteur RA, Carr EA, et al: Influence of tumor cell proliferation and sex-hormone receptors on effectiveness of radiation therapy for dogs with incompletely resected meningiomas. J Am Vet Med Assoc 216:701-707, 2000 42. Theon AP, Madewell BR, Shearn VI, et al: Prognostic factors associated with radiotherapy of squamous cell carcinoma of the nasal planum in cats. J Am Vet Med Assoc 206:991-996, 1995 43. Periosi R, Sarli G, Benazzi C, et al: Detection of proliferating cell nuclei antigen (PCNA) in canine and feline mammary tumors. J Comp Pathol 113:301-313, 1995 44. Funakoshi Y, Nakayama H, Nishimura R, et al: Cellular proliferation and telomerase activity in canine mammary tumors. Vet Pathol 37:177-183, 2000 45. Lohr CV, Teifke JP, Failing K, et al: Characteristics of proliferation state in canine mammary tumors by the standardized AgNOR method with postfixation and immunohistologic detection of Ki-67 and PCNA. Vet Pathol 34:212-221, 1997 46. Pena LL, Nieto AI, Perez-Alenza D, et al: Immunohistochemistry detection of Ki-67 and PCNA in canine mammary tumors: relationship to clinical and pathologic variables. J Vet Diagn Invest 10:237245, 1998 47. Nieto A, Pena L, Perez-Alenza MD, et al: Immunohistochemical detection of estrogen receptor alpha in canine mammary tumors: clinical and pathological associations and prognostic significance. Vet Pathol 37:239-247, 2000 48. Reels S, Tilmant K, Ducatelle R: PCNA and Ki-67 proliferation markers as criteria for prediction of clinical behavior of melanocytic tumors in cats and dogs. J Comp Pathol 121:13-24, 1999 DIAGNOSTIC PATHOLOGY FOR THE CANCER PATIENT
49. Laprie C, Abadie J, Amardeilh MF, et al: MIB-1 immunoreactivity correlates with biologic behavior in canine cutaneous melanoma. Vet Derrn 12:139-147, 2001 50. Viste JR, Myers SL, Singh B, et al: Feline mammary adenocarcinoma: Tumor size as a prognostic indicator. Can Vet J 43:33-37, 2002 51. Phillips BS, Kass PH, Naydan DK, et al: Apoptotic and proliferation indexes in canine tymphoma, J Vet Diagn Invest 12:111-117, 1999 52. FourneI-Fleury C, Magnol JP, Chabanne L, et al: Growth fractions in canine non-Hodgkin's lymphomas as determined in situ by the expression of the Ki-67 antigen. J Comp Pathol 117:62-72, 1997 53. Bolon B, Calderwood-Mays MB, Hall BJ: Characteristics of canine melanomas and comparison of histology and DNA ploidy to their biologic behavior. Vet Pathol 27:96-102, 1991 54. Fox MH, Armstrong LW, Withrow SJ, et al: Comparison of DNA aneuploidy of primary and metastatic spontaneous canine osteesarcomas. Cancer Res 50:6176-6178, 1990 55. Clemo FAS, DeNicola DB, Carlton WW, et al: Flow cytometric DNA ploidy analysis in canine transitional cell carcinoma of urinary bladders, Vet Pathol 31:207-215, 1994 56. Rutteman GR, Cornelisse CJ, Kijkshoorn JN, et al: Flow cytometric analysis DNA ploidy in canine mammary tumors. Cancer Res 48: 3411-3417, 1988 57. Madewell BR, Deitch AD, Higgins RJ, et al: DNA flow cytometric study of the hyperplastic and neoplastic canine prostate. Prostate 18:173-179, 1991 58. Minke JM, Cornelisse CJ, Stelwijk JA, et al: Flow cytometric DNA ploidy analysis of feline mammary tumors. Cancer Res 50:40034007, 1990 59. Hahn KA, Richarson RC, Hahn EA, et al: Diagnostic and prognostic importance of chromosomal aberrations identified in 61 dog with lymphosarcoma. Vet Pathol 31:528-540, 1994 60. Sprangler WL, Culbertson MR, Kass PH: Primary mesenchymal (non-angiomatous/non-lymphomatous) neoplasms occurring in the canine spleen: anatomic classification, immunohistochemistry, and mitotic activity correlated with patient survival. Vet Pathol 34:37-47, 1994 61. Sprangler WL, Kass PH: Pathologic and prognostic characteristics of splenomegaly in dogs due to fibrohistiocytic nodules: 98 cases. Vet Pathol 35:488-498, 1998 62. Langenback A, McManus PM, Hendrick MJ, et al: Sensitivity and specificity of methods of assessing the regional lymph nodes for evidence of metastasis in dogs and cats with solid tumors. J Am Vet Med Assoc 218:1424-1428, 2001 63. Wilcock BP, Yager JA, Zink MC: The morphology and behavior of feline cutaneous mastocytomas. Vet Pathol 23:320-324, 1986 64. Patnaik AK, Ehler WJ, MacEwen EG: Canine cutaneous mast cell tumor: Morphologic grading and survival time in 83 dogs. Vet Pathol 21:469-474, 1984 65, Kurzman ID, Gilbertson SR: Prognostic factors in canine mammary tumors. Sere Vet Med Surg 1:25-32, 1986 66. Weijer K, Head KW, Misdorp W, et al: Feline malignant mammary tumors I. Morphology and biology: Some comparisons with human and canine mammary carcinomas. J Natl Cancer Inst 49:1697-1704, 1972 67. Trojani M, Contesso G, Coindre JM, et al: Soft-tissue sarcomas of adults: study of pathological prognostic variables and definition of a histopathological grading system. Int J Cancer 33:37-42, 1984 68. Coindre JM, Trojani M, Contesso G, et al: Reproducibility of a histopathologic grading system for adult soft tissue sarcoma. Cancer 58:306-309, 1986 69. Bostock DE: Prognosis after surgical excision of canine melanomas. Vet Pathol 16:32-40, 1979 70. Wilcock BP, Peiffer RL: Morphology and behavior of primary ocular melanomas in 91 dogs. Vet Pathol 23:418-424, 1986 71. Valli VE, Norris A, Jacobs RM, et al: Pathology of canine bladder and urethral cancer and correlation with tumor progression and survival. J Comp Pathol 113:113-130, 1995 72. Straw RC, Le Couteur RA, Powers BE, et al: Multilobular osteochondrosarcoma of the canine skull: 16 cases (1978-1988). J Am Vet Med Assoc 195:1764-1769, 1989 73. Carter RF, Harris CK, Withrow SJ, et at: Chemotherapy of canine lymphoma with histopathological correlation: Doxirubicin alone compared to COP as first treatment regimen. J Am Anita Hosp Assoc 23:587-596, 1987
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74. Carter RF, Valli VEO, Lumsden JH: The cytology, histology and prevalence of cell types in canine lymphoma classified according to the National Cancer Institute working formulation. Can J Vet Res 50:154-164, 1986 75. Carpenter LG, Withrow SJ, Powers BE, et ah Squamous cell carci-
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noma of the tongue in 10 dogs. J Am Anim Hosp Assoc 29:17-24, 1993 76. Kirpenstein J, Kik M, Rutteman GR, et al: Prognostic significance of a new histologic grading system for canine osteosarcoma. Vet Pathol 39:240-246, 2002
SHELLEY JOY NEWMAN
It is necessary to have a cooperative relationship between the clinician and the pathologist, because information from both is critical for determining the best case management and favorable prognosis. The principle roles of the veterinary pathologist are to describe the submitted tissues, to determine the nature of the tumor, and to assess completeness of surgical margins. Recently, the Council of the American College of Veterinary Pathologists (ACVP) appointed an ad hoc committee on oncology to evaluate oncologists' needs by polling members of the Veterinary Cancer Society (VCS). The committee's vision was to review morphologic, immunologic, and molecular techniques so that veterinary pathologists could meet the current and future diagnostic and prognostic needs of oncologic veterinarians. The factors determined to be most important to the VCS members in selecting a diagnostic pathology provider included, in order of importance: (1) reliability of results; (2) short turnaround time; (3) easy access for consultation; (4) rapport with pathologist; (5) convenience of service; and (6) cost of service. This manuscript will attempt to review improved morphologic, immunologic, and molecular techniques to provide for the future diagnostic and prognostic needs of oncologic veterinarians. © 2003 Elsevier Inc. All rights reserved.
here is a strong cooperative relationship required between
T the clinician and the pathologist, because information from both is critical for determining the best case management and prognosis. ~ The principle roles of the veterinary pathologist have been determined to be description of the submitted tissues, determination of the nature of the tumor, and assessment of completeness of surgical margins. 2 Determining prognosis may be more difficult because the pathologist often is involved relatively late in the course of the disease and rarely has all the relevant information to clearly summarize the case. x,3 Thus, the clinician's role is to interpret all the data and determine the therapeutic plan accordingly. Ensuring the reliability of results is a necessary goal of any quality diagnostic service, but is influenced by numerous variables, including those involving the clinician, the laboratory, and the pathologist. Submission of unlabeled containers, or containers with multiple unidentified dermal biopsy samples and incomplete or absent patient information forms are means by which clinicians contribute to unreliable results. Additionally, improper fixation ratios (less than 1:10 in 10% buffered formalin) produce tissue artifact. 4 Thus, for large specimens, the clinician should select representative samples for submission while storing the formalin-fixed specimen at the clinic or,
From the Animal Medical Center, New York, NY. Address reprint requests to Dr. Shelley Newman, The Animal Medical Center, 510 East 62nd Street, New York NY, 10021-8314. © 2003 Elsevier Inc. All rights reserved. 1096-2867/03/1802-0017530.00/0 doi:10.1053/svms.2003.36633
following complete fixation, submit the entire specimen in a smaller container. 1,5 Bouins fixative may provide superior fixation for reproductive or ocular specimens. It may be necessary for a clinician to maintain a variety of sizes of containers as well as a large container of formalin for large specimens, such as spleens or limbs. Because extent and appropriateness of tissue sampling are also critical to an accurate diagnosis, at least 3 representative sections (rather than large focal areas of hemorrhage, inflammation, or necrosis), including some that border normal tissue, are recommended, because this assists in determining the degree of invasiveness of the tumor. 1,5,6 If excisional biopsies are submitted, then margins of interest should be marked and an interpretive summary included. 1 Methods of marking surgical margins include preferential inking (India ink or fluorescent surgical marking ink), suturing, or staplingY r If the entire sample is not submitted for pathology, representative labeled margins must be sent.S Confusion occurs with inking if margins are contaminated during processing and interpreted as surgical margins. 4 The following laboratory-related factors can confound the reliability of results: loss tof samples, freezing or leakage of samples such that inadequate fixation results in tissue artifact, incorrect trimming, improper tissue orientation, disintegration of small samples, shatter artifact in osseous or mineralized tissues, collapse of tissues, ie, eye, and mislabeling of blocks or slides during processing. 1.9 Ensuring and maintaining competency of pathologists is a difficulty, as in all professions that revolve around subjective opinion. Controversies exist most commonly in diagnosis, grade, or completeness of margins. 3 For diagnosis, this may be in part due to the variable use of older terminology, referral to human pathologists untrained in veterinary pathology, difficulty in distinguishing benign tumors from well-differentiated malignant tumors, and the complications of marked inflammation and granulation tissue response in tumor diagnosis. 1 Resectioning available tissues and performing a series of special light microscopic or immunohistochemical stains may be useful in such cases. 5 Additionally, seeking multiple opinions from pathologists at larger facilities or those with specialty expertise can minimize these concerns. 5 The advent of telemedicine may allow for transfer of images via the Internet for consultation with others at a distance from the primary laboratory. All pathologists should strive to deliver a biopsy report that contains a detailed histologic description, an assessment of completeness of excision, a final diagnosis, and a grade, if applicable. 4 As determined by the ACVP ad hoc committee's oncologist survey, the components of greatest importance within a pathology report are: l) surgical margins; 2) diagnosis; 3) grade; 4) histologic description; 5) mitotic index; and 6) pathologist's opinion of prognosis)
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1;39
Surgical Margins The best way for a pathologist to assess surgical margins is to use visual indicators such as ink, in a variety of colors, to allow for proper tissue orientation and interpretation. Margin assessment is still subject to interpretive differences, because it is difficult to transmit 3-dimensional margins into a 2-dimensional light microscopic plane, and to conclude that all margins are free of infiltrating cells based on evaluation of a 5-/xm thick section. 4 Sometimes the clinician is better off relying on knowledge of likely clinical behavior rather than on a literal interpretation of the surgical margins. 4 A 10% recurrence rate for human soft-tissue sarcomas with complete excision is documented, although the recurrence rate is higher in veterinary medicine. ~° Completeness of margins can be described as radical, wide, marginal, or dirty. Dirty margins occur when tumor cells extend to the surgical margin. 1 Marginal (clean but close) margins occur when there is less than 1 cm of normal tissue present around the neoplasm, s Wide margins are seen when 1 to 3 cm of normal tissue surround the tumor. ~ Radical margins are attained when the entire compartment is removed, ie, amputation. 1
Diagnosis Once a diagnosis of neoplasia---defined as a disturbance of growth characterized by excessive, uncontrolled proliferation of cells---is determined, then the origin and nature of the tumor must be evaluated. Benign tumors typically resemble the normal tissue of origin, grow slowly by expansion and compression, and fail to metastasize. Life-threatening effects may still occur when these benign tumors are located in surgically compromised locations, are predisposed to rupture and hemorrhage, and are secreting hormones that affect physiologic function.1 Cellular features of benign masses include low nuclear/ cytoplasmic ratio, nuclei of uniform size and shape, and a low mitotic rate. 1,3,8,H In contrast, most malignant tumors grow rapidly and invade the surrounding tissues. Invasion may involve infiltrative cell aggregates, invasion of vascular or lymphatic spaces, or induction of a desmoplastic response as seen by excess fibrous connective tissue reaction. 1 Features of malignant neoplasms are poor cellular differentiation, high cellularity, high nuclear/cytoplasmic ratio, variation in nuclear (anisokaryosis) and cytoplasmic size (anisocytosis), variation in nuclear shape, large nucleoli, multiple nucleoli, increased necrosis, abnormal mitotic figures, and a high mitotic rate. ~ Neoplasms originate from epithelial or mesenchymal tissue. 2 Benign mesenchymal tumors originating from connective tissue, striated or smooth muscle, endothelial cells, or meninges are designated with the suffix "oma." Those of malignant origin carry the corresponding suffix "sarcoma." Similarly, benign epithelial tumors originating from glandular tissue carry the suffix "adenoma," and those that originate from solid epithelium, "papilloma." The malignant epithelial tumors are designated "adenocarcinoma" or "carcinoma," respectively. 8 Teratomas are exceptions because they are neoplasms composed of at least two different embryonic germ layers. Two neoplastic tissues can often be seen in mixed tumors in which glandular cells and myoepithelial cells proliferate, such as in mammary or salivary tumors. Additionally, there are some tumors in which histologic features do not necessarily correlate with behavior;
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canine histiocytoma, 1 infiltrative lipoma, t2 and histologically low-grade, biologically high-grade fibrosarcoma of the canine head. 13 A recent report determined that classic histologically benign canine choroidal melanomas can exhibit unexpected malignant behavior, as can conjunctival melanomas. I<~5 Approximately 90% of human cancer cases can be diagnosed by light microscopy alone. 1,~6,~r The remaining 10% require special procedures. ~ Special light microscopic stains such as toluidine blue, periodic acid Schiff PAS, alcian blue, or Masson Trichrome can identify mast-cell granules, mncopolysaccharide, ground substance, and collagen fibrils, respectively. More recently, immunohistochemistry has been more widely used to determine tissue of origin by identifying intermediate filaments and other structural cytoplasmic proteins. This procedure works on the premise that primary antibodies recognize specific cell components and then the secondary antibody is directed against the primary antibody. This complex becomes visible because the secondary antibody is linked to peroxidase or avidin biotin peroxidase complexes that catalyze a reaction to precipitate a dye on the complex. 16,18Heterogeneous tumorcell populations can yield inconsistencies in protein expression and staining, and excessive fixation can create false results by cross- linking the antibody. 1 Thus, it is best to perform panels of antibodies to obtain accurate and useful information, but even then, staining should be used only as a guide and not as a definitive indicator of neoplastic cell type. 16 Additionally, immunohistochemistry does not distinguish between benign and malignant cells. 1 The two most utilized markers for anaplastic tumors include cytokeratin for epithelial origin and vimentin for mesenchymal origin. Only rare tumors, such as mesotheliomas and synovial cell sarcomas, can stain for both markers. xg,x° Chromagranin A ig a useful marker for pancreatic tumors of neuroendocrine origin and their metastases, xx,x2Other endocrine tumors can be characterized by a series of primary products such as thyroglobin in thyroid carcinomas, calcitonin in medullary carcinomas, and thyroid transcription factor-1 in a proportion of thyroid follicular carcinomas. 23 Melan A is more specific but less sensitive than S100 for differentiating melanoma from pigmented basal cell tumor in cats, the latter of which would also be cytokeratin positive. 24 The most useful marker to determine dendritic cell origin for histiocytic sarcoma in dogs is positivity for CD18 and negativity for CD3 and CD79a. 25 Lastly, some neoplasms may revert to expressing fetal antigens, as is seen in a proportion of renal cell carcinomas that express cyclo-oxygenase (COX). a6 The accuracy of predicting prognosis, malignancy, or response to therapeutic modalities is significantly increased by the results of special procedures, and pathologists are in the best position to recommend these tests. 4 Immunohistochemistry staining techniques for proliferation markers or altered gene products, and silver staining (Ag) for nucleolar organizer regions (NORs) is fast progressing. In malignant cells, NORs desegregate from the nucleoli and disperse throughout the nucleus to permit RNA transcription.5 The size and number noted are related to nuclear activity. 5 An increase in number and area of AgNORs is correlated with cell proliferation, a The stained complex corresponds to the binding sites of RNA polymerase 1 and associated proteins regulating transcription and post-translational modification of ribosomal RNA. 8 Automated image analysis can minimize observer bias and is less time consuming and tedious than manual counting methods. 5 AgNORs have
SHELLEY JOY NEWMAN
been determined to be a valuable prognostic marker for the majority of tumors studied. Additionally, prognostic information can result from utilizing monoclonal antibodies against epitopes of antigens expressed by proliferating cells# Examples are proliferating cell nuclear antigen (PCNA) and MIB-1 antibody directed against Ki67, which are both nonhistone proteins detected in varying amounts during several phases of the cell cycle. 5 Canine malignant lymphomas with a longer disease-free interval and survival time correlated with larger mean AgNOR area, larger maximal AgNOR area, a larger total AgNOR area, shorter distance between two AgNORs, a smaller AgNOR area to nucleus ratio, and a smaller number of AgNORs per nucleus. 2r Similarly, AgNOR counts correlated inversely with response to therapy in an additional lymphoma study. 28 Canine transmissible venereal tumor with high AgNOR counts had poor chemotherapy repsonses. 29 Metastatic canine seminomas had higher AgNOR counts compared with nonmetastatic forms. 3° Canine perianal tumors had higher AgNOR counts in the malignant variantsY AgNOR counts correlated with histologic grade and prognosis following surgery in canine mammary tumors, 32 and in one study, AgNOR index for 1-year survival was the most significant parameter influencing survival. 33 Additionally, higher AgNOR scores were seen in malignant f o r r n s , 34,35 but no difference was noted between metastatic and nonmetastatic forms. 34 In feline mammary tumors, high AgNOR count was agsociated with shorter postoperative survival times, 36 but AgNOR cluster size did not correlate to mitotic index, tumor stage, nuclear atypia, or histologic appearance. 3r Mast cell tumors had a poorer prognosis if AgNOR and PCNA counts were high and c-kit was identified. 38-4° Both canine meningioma and feline squamous cell carcinoma with high PCNA levels were associated with poor responses to radiation therapy. 41,42 In canine and feline mammary tumors, high PCNA is associated with malignant t u m o r s , "~3,44 and predicted local tumor recurrence following surgery. 45 Canine mammary tumors with high Ki67 are associated with malignancy: metastasis, low disease-free survival rate, poor prognosis, and risk of death. B3,46 Ki67 is inversely correlated with estrogen receptor alpha scores. 47 Canine and feline melanotic tumors demonstrated higher Ki67 in malignant than in benign tumors. 48,49 Lack of correlation between Ki67 and postsurgery survival suggests that this marker alone is not sufficient to determine the correct prognosis in feline mammary carcinomas, and the histologic grading system actually has a higher predictive value. 5° In canine lymphoma, Ki67 correlated with histologic grade, 5~ and with prolonged first remission duration after chemotherapy. 52 Flow cytometry can be utilized to determine ploidy (the nuclear DNA content of cells), which can aid in distinguishing malignant tumors from other lesions. Aneuploidy is most associated with neoplastic transformation, and has been documented with these tumors: canine malignant melanomas, 53 osteosarcomas, 54 transitional cell carcinomas, 55 canine m a m m a r y adenocarcinomas, 56 prostate carcinomas, 5r and a minority of feline m a m m a r y adenocarcinomas. 58 There has been little correlation between ploidy and histology, survival time, or clinical characteristics for transitional cell carcinoma. 55 Presence of chromosomal aberrations, namely trisomy of chromosome 13 in canine lymphoma, produced a significantly longer first remission length and survival time. 59
DIAGNOSTIC PATHOLOGY FOR THE CANCER PATIENT
Grade The widespread usage of grading systems is fraught with difficulty because there are a variety of schemes, some of which are unfounded, and many of which utilize subjective terms such as "poorly," "moderately," or "well differentiated." Additionally, some tumors are not amenable to grading schemes. Most grading schemes are based on mitotic rate, degree of cellular differentiation, cellular or nuclear pleomorphism, and percentage of necrosis. 1 Other important features include overall cellularity, degree of invasiveness, amount of inflammation, nucleolar size, amount of stromal reaction, and lymphoid response, s.s Current grading schemes vary and may focus on one criterion, such as mitotic index, and result in low, medium, or high-grade designations, whereas others are based on a combination of criterion. 8 Only mitotic index and amount of necrosis are considered objective. I The behavior of nonangiomatous/nonlymphomatous splenic sarcomas was found to be closely correlated with mitotic index, where those with an index greater than 9 had a shorter survival time. 6° Similarly, mitotic index in combination with lymphoid:fibrohistiocytic proportion in splenic nodules were the anatomic features most predictive of postsplenectomy mortality. 61 High mitotic index and a low lymphoid:fibrohistiocytic proportions indicated a greater prObability of higher short-term mortality. 61 Frequently, cellular morphology is the most accurate predictor of behavior of a neoplasm. 8 Individual tumors may have specific associations between prognosis and morphology, and the latter is utilized in proven morphological grading schemes, s Ultimately, the grade is important for prognosis, but also for determination of the need for adjuvant therapy. 1 Analysis of lymph-node tissue can also confirm metastatic disease, which affects overall prognosis, substantiates grade, and determines stage. Fine-needle aspiration was determined to be a sensitive and specific method of evaluating the regional lymph nodes in dogs and cats because it correlated well with lymph-node histology. 62 Carcinomas showed a higher incidence of lymphnode metastases than sarcomas. 62 Grading of tumors is becoming increasingly important because of the behavior of some tumors being shown to be dependent on grade. 1 Examples where grade or histologic features predict outcome include the following: canine and feline mastcell t u m o r , 63,64 canine and feline mammary gland adenocarcinoma, 65,66 canine soft-tissue s a r c o m a s , 6r,68 canine cutaneous and ocular melanoma, 69,r° canine splenic nonhematogenous/ nonlymphomatous sarcoma, 6° synovial cell sarcoma, 2° transitional cell carcinoma, rl multilobular osteochondrosarcoma, r2 lymphoma, r3,r4 and squamous cell carcinoma of the tongue, r5 The profession agrees that improved grading schemes are required for these important tumors: lymphomas, lymphoid leukemias, nonlymphoid leukemias, histiocytic tumors, soft-tissue sarcomas, mammary tumors, and mast-cell tumors. 3 Such advanced grading schemes might incorporate AgNOR and proliferation cell marker counts. The pathologist must stay abreast of the literature regarding current grading schemes, s The most recently published histologic grading scheme is for canine osteosarcoma, and is based on histologic criterion such as cellular pleomorphism, mitoses, amount of matrix, percentage of tumor cells, and amount of necrosis. 76 Many of the more accepted schemes have been adapted from human medicine. ~ The grading system for canine soft-tissue
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sarcomas is adapted from that in humans, and tends to rely more on grade than the cell of origin. ~,6r,68 Tumors are scored for degree of differentiation, mitotic rate, and necrosis, and an overall grade assigned following addition of the numeric scores. ~,6r,68 Grade 1 encompasses a numeric score of 3 or 4, grade 2 a score of 5 or 6, and grade 3 a score of 7, 8, or 9.11,67,68 It was found that the combination of these 3 factors was necessary to result in useful prognostic information. 6r Histologic grade was the most important prognostic factor for human soft-tissue sarcomas, 6r and the grading system was reproducible among human pathologists. 68 This is suspected to be similar in veterinary medicine. In addition to grading, staging systems are intended to determine prognosis, to aid in planning treatment, and to evaluate the success of treatment regimes, s,ls The TMN system developed by the World Health Organization classifies tumors by local (T), regional (N), and metastatic (M) characteristics, s Although TO indicates no evidence of neoplasia, T1 to T4 are associated with increasing size of the primary mass. NO means lymph nodes are not involved, but N1 to N3 is utilized for increasing nodal involvement. M0 indicates an absence of metastases, which are otherwise recorded as M1 or M2. These values should be taken into consideration along with all other data to assist with determining prognosis and treatment decisions. 8
Histologic Description An ideal report should contain a detailed microscopic description, a final diagnosis, and a grade, if applicable. 4 Often, the description will assist in determining the limits of a complete clinical evaluation, proper treatment options, and an accurate prognosis. 4
Mitotic Index As an isolated result, this is not considered important but can be an integral of accepted grading schemes. Mitotic index is the most utilized criterion to indicate degree of proliferation, and in some tumors, such as melanoma and other sarcomas, this has been determined to be the best predictor of survival and/or response to treatment, although this does not hold true for histiocytomas or transmissible venereal tumors. 8
Prognosis Many feel that pathologists should not make blanket statements about prognosis because they rarely have all the clinically relevant information to do so. Additionally, much of the prognostic data are extrapolated from humans or from a relatively small body of survival study data for individual tumors in the veterinary literature. Ultimately, we should develop classification schemes using morphologic, immunologic, and cytochemical characteristics that better predict biologic behavior and response to therapy for tumors of veterinary importance.
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