Oncology–Original Article

Veterinary Pathology
2015, Vol. 52(2) 238-249
Prognostic Value of Histologic Grading ª The Author(s) 2014
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for Feline Mammary Carcinoma: DOI: 10.1177/0300985814543198
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A Retrospective Survival Analysis

S. W. Mills1*, K. M. Musil1*, J. L. Davies2, S. Hendrick1, C. Duncan3,

M. L. Jackson1, B. Kidney1, H. Philibert1, B. K. Wobeser1,
and E. Simko1

Abstract
Feline mammary carcinoma is highly malignant and generally associated with a poor prognosis, although studies suggest the range
of survival times in affected cats is broad. Histologic grading of these tumors is achieved using the Elston and Ellis system, originally
developed for human breast cancer. In cats, however, classification using this method has variable prognostic value. Therefore,
objectives of this study were (1) to evaluate the Elston and Ellis grading system for feline mammary carcinoma in a predominantly
spayed population and (2) to determine whether modification of this system or development of a novel system improved the
prognostic value of histologic grading. Survey data and histologic features for 108 carcinomas from 97 cats were analyzed with
respect to overall survival. Elston and Ellis grading failed to correlate significantly with overall survival. Using multivariable analysis,
lymphovascular invasion, nuclear form, and mitotic count each demonstrated independent prognostic significance (P ¼ .008,
<.001, and .004, respectively). Modifications of the Elston and Ellis system and a novel grading system were proposed based on
these results; all showed significant correlation with overall survival (P < .001). Median survival times were 27, 29, or 31 months
for grade I; 14, 12, or 14 months for grade II; and 13, 5, or 8 months for grade III carcinomas using the mitotic-modified Elston and
Ellis, the revised Elston and Ellis, or the novel grading system, respectively. Based on this retrospective study, adoption of the
species-specific systems as proposed here may improve the prognostic value of histologic grading for feline mammary carcinoma.

Keywords
feline mammary carcinoma, tumor histology, grading, prognosis, retrospective postoperative survival, Elston and Ellis

Feline mammary chain tumors are encountered routinely in pri- tubule formation, nuclear pleomorphism, and mitotic count)
mary veterinary practice, accounting for 17% of neoplasms in are scored and added together to produce a grade, which
female cats.15 Reported malignancy rates generally range then correlates with degree of malignancy and prog-
between 80% and 90%,9,15 with fibroadenoma, fibroadenoma- nosis.8,30,31 Use of this grading system by veterinary pathol-
tous hyperplasia, or duct ectasia being the most commonly ogists is based primarily on evidence suggesting that feline
encountered benign, dysplastic, or nonneoplastic masses, mammary carcinoma represents a suitable model for human
respectively.9 The etiology is unknown but almost certainly
multifactorial; reported risk factors include sex,12 breed,11,14
age,38 hormone exposure,21 and reproductive status.26
Although feline mammary carcinomas tend to be biologically *
Indicates equal contribution
aggressive, survival times can vary significantly.19,29,36 Prog- 1
Western College of Veterinary Medicine, University of Saskatchewan,
nosis in affected cats has been shown previously to be influenced Saskatoon, SK, Canada
2
by tumor diameter,14,16,36,38,39 World Health Organization Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
3
College of Veterinary Medicine and Biomedical Sciences, Colorado State
(WHO) stage and modified WHO stage,14,34 extent of sur- University, Fort Collins, CO, USA
gery,16,25 and histologic grade.4,34 The Elston and Ellis (EE) his-
tologic grading system (also known as the Nottingham Supplemental material for this article is available on the Veterinary Pathology
Grading System) has been widely adopted by veterinary website at http://vet.sagepub.com/supplemental.
investigators for the grading of feline mammary carci-
Corresponding Author:
noma.2,4,19,28,32–34,40 This system represents the ‘‘gold stan- S. Mills, Western College of Veterinary Medicine, University of Saskatchewan,
dard’’ in assessment of invasive human breast cancer, 52 Campus Drive, Saskatoon, SK, Canada.
whereby distinct histopathologic features (ie, percentage Email: [email protected]
Mills et al 239

breast cancer, particularly the more aggressive estrogen- greater than 3 cm in diameter with or without evidence of
negative carcinomas.2,22,39,41 regional metastases. Stage IV included any cat with evidence
Castagnaro et al4 found the EE grading system to have of distant metastases regardless of tumor size or regional
good predictive value for well-differentiated (grade I) and metastases.
poorly differentiated (grade III) carcinomas. The same grad-
ing method was found to have high predictive value in
queens with invasive carcinomas by Millanta et al.19 More
Inclusion/Exclusion Criteria
recently, Seixas et al34 found histologic grade determined This study included only primary mammary tumors from
using the EE grading system to be an independent prognos- female cats (spayed or intact) with complete survey data, a spe-
tic factor for middle-aged to elderly queens with mammary cimen of adequate quality, a confirmed histologic diagnosis of
carcinoma. These studies present compelling preliminary invasive mammary carcinoma, and a WHO clinical stage of I,
evidence in support of histologic grading using the EE grad- II, or III. Except in cases where death or euthanasia occurred
ing system as a predictor of overall survival (OS) time in and was determined or suspected to be tumor related, at least
unspayed female cats. 1 year of follow-up information was required for inclusion.
Matos et al18 recently called attention to the need for a Any cats receiving treatment beyond surgical excision of the
standardized grading system for feline mammary gland car- tumor (ie, drug or radiation therapy) were excluded. Noninfil-
cinomas, where early detection, reliable histologic charac- trating (in situ) carcinomas were excluded. A tumor was con-
terization, and aggressive treatment may have a significant sidered primary when there was no prior history of mammary
influence on survival times. The aim of the current study neoplasia indicated in the pathology report or survey data.
was to (1) determine whether histologic grade using the Every effort was made to confirm mammary origin. In cases
EE grading system is a reliable prognostic indicator for a that initially presented with multiple mammary masses, all sub-
predominantly spayed, North American population of mitted tumors were evaluated independently. A total of 97 cats
female cats with mammary carcinoma and (2) determine fulfilled the case definition, encompassing 108 tumors. Of
whether modification of the EE grading system and/or those, 88 (81%) were single tumor submissions, while 20
development of a novel grading system would improve the (19%) were multiple tumor submissions from the same animal.
prognostic value of routine histologic examination of feline
mammary carcinoma.
Processing of Tissues
Archived, paraffin-embedded tissues were retrieved, sectioned
Materials and Methods (4 mm), and stained with hematoxylin and eosin (HE). When it
was necessary to confirm intravascular invasion, vascular
Case Origin and Data Collection endothelial cells were highlighted by immunohistochemical
Cases were drawn from 834 surgical biopsy specimens of staining for Von Willebrand factor carried out on paraffin-
mammary tumors originating from 789 domestic cats and sub- embedded sections using a commercial staining platform
mitted to either Veterinary Diagnostic Services (later Prairie (Benchmark staining platform; Ventana Medical Systems,
Diagnostic Services, Inc) at the University of Saskatchewan Tucson, AZ) and a streptavidin-biotin detection system (BMK
between 1989 and 2011 or to the Veterinary Diagnostic iVIEW DAB Paraffin detection kit; Ventana Medical Sys-
Laboratories at Colorado State University between 2005 and tems). The primary antibody (polyclonal rabbit anti–human
2010. Use of animal tissues in this study was approved by the Von Willebrand factor; Dako Canada, Inc, Mississauga, ON,
University Committee on Animal Care and Supply at the Uni- Canada) was applied for 32 minutes at a dilution of 1:1000.
versity of Saskatchewan. A total of 329 surveys were distrib-
uted to referring clinics to obtain follow-up information based
on the original medical case file. Partial or complete informa- Histologic Assessment Criteria
tion on 225 cases comprising 255 excisional biopsy speci- Histologic slides were reviewed independently by 2 authors
mens (tumors) was obtained. The survey collected the (S.W.M. and K.M.M.), with direct assistance from two ACVP
patient signalment; size, location, appearance, and duration board-certified veterinary pathologists (J.L.D. and E.S.).
of mammary tumor(s); presence and location of lymph node Assessors were blinded to the clinicopathologic (survey) data
and/or distant metastasis at initial diagnosis; presence and and clinical outcome; all discordant results were reviewed by
timing of local/regional recurrence or metastases; and the tim- S.W.M. and K.M.M. with a multiheaded microscope to reach
ing and nature of the eventual outcome. All cases were staged consensus. When more than 1 section was available for exam-
according to the modified WHO staging system.15 Briefly, ination, only the one containing the largest portion of the tumor
stage I and stage II included cats with primary tumors less was selected for evaluation. The only exception was assess-
than 2 cm and 2 to 3 cm in diameter, respectively, with no evi- ment of lymphovascular invasion, where all available sections
dence of regional or distant metastases. Stage III included cats were screened. All tumors were initially classified according to
with primary tumors less than 2 cm or 2 to 3 cm in diameter the WHO subtypes described for malignant feline mammary
with evidence of regional metastases or cats with tumors carcinomas.20 The current study included tubulopapillary,
240 Veterinary Pathology 52(2)

solid, cribriform, and squamous cell carcinomas, with most were evaluated in the least differentiated and/or most invasive
demonstrating more than 1 growth pattern. In these instances, portion of the tumor, and the number of nuclei exhibiting abnor-
the pattern comprising the largest relative portion of the tumor mal form was estimated relative to the total number of nuclei
determined the subtype. within a given field and expressed as a percentage. Subcate-
Tumor characteristics evaluated included percentage tubule gories (5%, 6%–25%, or >25% abnormal) were then assigned.
formation, necrosis, squamous differentiation, inflammation, Mitotic figures were evaluated according to Elston and Ellis.8
lymphovascular invasion, stromal response, chromatin vesicu- Briefly, mitotic figures were counted in 10 consecutive fields
lation, nucleolar morphology, anisokaryosis, nuclear size, at the periphery of the tumor in the areas of highest proliferative
nuclear form, nuclear pleomorphism, and mitotic count. Per- activity. Microscope field diameter was maintained at 0.53 mm
centage tubule formation was evaluated according to Elston for all observations to standardize counts.1 Care was taken
and Ellis.8 Briefly, the proportion of neoplastic cells within a to exclude apoptotic, pyknotic, or otherwise hyperchromatic
given section exhibiting discrete tubule formation relative to nuclei, only including those that had definitively entered pro-
the total tumor mass was expressed semi-quantitatively as a phase or were in metaphase, anaphase, or telophase. In certain
percentage. Necrosis and squamous differentiation were scored cases where tumor heterogeneity and high mitotic rate caused
in similar fashion. Changes consistent with squamous differen- mitotic count to differ by more than 20% between 2 observers,
tiation included at least 2 of the following: decreased N:C ratio, they were repeated independently until acceptable concordance
increased cytoplasmic eosinophilia, increased cell size, and was attained. A correlation coefficient (Pearson’s R value) of
increased angularity of cell margin. Inflammation was classi- 0.954 was obtained overall.
fied subjectively as follows: (1) absent or very mild, (2) predo-
minantly lymphoplasmacytic, or (3) neutrophilic or
pleocellular. A tumor was positive for lymphovascular inva-
Histologic Grading
sion if neoplastic emboli were clearly seen within a continuous, Initially, tumors were graded according to the EE grading sys-
endothelium-lined vessel lumen. In cases where the assess- tem described for human breast cancer (Table 1).8 In this sys-
ment of lymphovascular invasion was equivocal or consensus tem, carcinomas are scored according to 3 criteria: percentage
could not be reached, additional sections were stained for Von tubule formation, degree of nuclear pleomorphism, and mitotic
Willebrand factor to highlight vascular endothelium and a count. Briefly, the degree of tubule formation is assessed sub-
definitive classification made. Stromal response encompassed jectively on low power and expressed as a percentage. When
desmoplasia, myofibroblastic/myoepithelial hyperplasia, or a greater than 75% of the tumor parenchyma exhibits tubule for-
combination of both; it was often not possible to definitively mation, 1 point is assigned. Tubule formation between 10% and
discriminate between these processes in HE-stained tissue 75% is given a score of 2 points, and 3 points are assigned when
sections. The stromal response was graded as normal (mild- <10% of the tumor parenchyma displays tubule formation.
moderate) or marked when limited primarily to the periphery Nuclear pleomorphism scoring is conducted at high power
of the tumor, or intratumoral when it dissected through the (40) in the least differentiated and/or most invasive portion
tumor in a trabecular fashion. Assessment of neoplastic cell of the tumor, typically along the periphery. Tumor cell nuclei
nuclei was limited to the least differentiated and/or most inva- that display uniform chromatin distribution with only mild var-
sive portions of the tumor in areas lacking appreciable squamous iation in size, shape, and contour relative to normal nuclei are
differentiation to reduce bias. Normal nuclear morphology was assigned 1 point. Enlarged nuclei with more vesicular chroma-
defined as that which may be found in normal mammary tubular tin and moderate variation in size and shape are given 2 points.
epithelium. Subcategories for chromatin vesiculation, nucleolar Finally, 3 points are assigned when tumor cell nuclei exhibit
morphology, anisokaryosis, nuclear size, and nuclear form were marked vesiculation as well as marked variation in size and
developed and assessed subjectively based on the range of shape. Nucleoli are very often present in the latter 2 cases; mul-
morphology observed in all specimens. Chromatin vesiculation tiple nucleoli or nucleolar atypia favor category 3.
evaluated the predominance of euchromatin within the nuclear Mitotic counts were grouped as follows: a total of 0 to 8
envelope as evidenced by areas of pallor or clear space. Cate- mitotic figures in 10 high-power fields (40) was given 1
gories included (1) none or mildly vesiculated, (2) moderately point, 9 to 16 was given 2 points, and >17 was given 3 points.
vesiculated, or (3) markedly vesiculated. Nucleoli were charac- The score for each category was summed, and the total score
terized as (1) indistinct, (2) small and prominent, or (3) large, for each tumor corresponded to a predetermined grade (I, II,
multiple, or abnormally shaped. Anisokaryosis and nuclear size or III), indicating well-differentiated, moderately differen-
were expressed relative to adjacent tumor cells and nuclei in nor- tiated, or poorly differentiated carcinomas, respectively.
mal mammary epithelium, respectively. The nuclear form Three new grading systems were designed, applied to our
assessment derived from a high-power (40–60 objective), sub- data, and analyzed with respect to OS. First, in the mitotic-
jective evaluation of nuclear shape independent of other nuclear modified Elston and Ellis (MMEE) grading system (Table 1),
features or artifactual changes (Figs. 1–4, Suppl. Figs. 1–6). range subcategories within the mitotic count category of the
Deviations from a smooth nuclear contour and round or oval EE grading system were modified to better accommodate the
shape such as corrugation, angularity, clefting (indentation), or wide range and high magnitude of mitotic counts observed
overtly ameboid shape were considered abnormal. Several fields within the tumors we evaluated. Cutoff values between mitotic
Mills et al 241

Figures 1–4. Mammary gland; cat, mammary carcinoma. Hematoxylin and eosin. Figure 1. Example of low nuclear form score (5% abnor-
mal). Figure 2. Higher magnification of Fig. 1. Figure 3. Example of high nuclear form score (>25% abnormally shaped nuclei). Figure 4. Higher
magnification of Fig. 3. Examples of abnormal nuclear form are frequently noted (eg, deviations from a smooth nuclear contour and round or oval
shape such as corrugation, angularity, clefting, indentation, or overtly ameboid shape; arrowheads).

count subcategories were derived directly from tertile bound- invasion together with less than or equal to 5% abnormal
aries from our results. Next, with the revised Elston and Ellis nuclear form and a mitotic count less than or equal to 62 in
(REE) grading system (Table 1), the EE grading system was 10 high-power fields corresponded to grade I (low-grade carci-
modified further to include nuclear form scoring and lympho- noma). The presence of any one of lymphovascular invasion,
vascular invasion. Nuclear pleomorphism scoring was greater than 5% abnormal nuclear form, or a cumulative mitotic
removed, and an additional point was added to the grading count greater than 62 yielded grade II (intermediate-grade car-
chart to accommodate the addition of lymphovascular invasion. cinoma). Finally, if any 2 or all 3 of the aforementioned fea-
Scoring and grading with the MMEE and REE grading systems tures were present, grade III (high-grade carcinoma) was
was otherwise achieved in the same manner as described above assigned.
for EE grading. Finally, a novel grading system was developed
directly from the Cox proportional hazards analysis (Table 2).
Hazard ratios were assigned to the presence of the independent Statistical Analysis
prognostic factors (lymphovascular invasion, nuclear form, and Analyses were conducted using Prism (GraphPad Software,
mitotic count) using coefficients from the model and divided La Jolla, CA) and Stata (StataCorp LP, College Station TX)
into grades. Mitotic count subcategories were derived directly statistical software packages. Normality was assessed with the
from the median mitotic count observed in our results. Using D’Agostino and Pearson normality test. To analyze the rela-
the novel grading system, the absence of lymphovascular tionship between individual survey or histologic criteria/
242 Veterinary Pathology 52(2)

Table 1. Elston and Ellis (EE), Mitotic-Modified Elston and Ellis (MMEE), and Revised Elston and Ellis (REE) Grading Systems for Evaluation of
Invasive Mammary Carcinoma in Female Cats.

Applicable Grading System Histologic Feature Score

EE, MMEE, REE Tubule formation

Comprises a majority of the tumor (>75%) 1
Present to a moderate degree (10%–75%) 2
Little or none present (<10%) 3
EE, MMEE Nuclear pleomorphism
Small, regular, uniform nuclei 1
Moderately increased size, vesiculation, and variability 2
Vesicular chromatin with marked variation in size and shape 3
EE, MMEE, REE Mitotic counta
EE MMEE, REE
0–8 0–50 1
9–16 51–70 2
17 71 3
REE only Lymphovascular invasion
Absent 0
Present 1
REE only Nuclear formb
5% abnormal 1
6%–25% abnormal 2
>25% abnormal 3

Point Total Grade Comment

3-5 I Well differentiated

6-7 II Moderately differentiated
8-9 or 8-10 (REE) III Poorly differentiated
a
Cumulative number of mitoses in 10 consecutive fields in the most mitotically active area with a microscope field diameter of 0.53 mm (40 objective).
b
Abnormal nuclear form includes any deviation from smooth nuclear contour or round/oval nuclear shape such as clefting, angularity, corrugation, or ameboid
morphology assessed at high power (40–60 objective) in the least differentiated and/or most invasive portions of the tumor (Figs. 1–4). The number of nuclei
exhibiting the abnormal nuclear form is estimated and expressed as a percentage of the total number of nuclei within any given field.

grading and OS, Kaplan-Meier curves were generated and and 1 Devon Rex (1%). Ovariectomy or ovariohysterectomy
compared using log rank or Gehan-Breslow-Wilcoxon tests. (spaying) had been performed previously in 82 cats (84.7%),
A Cox proportional hazards model was used to evaluate the while 15 (15.3%) were intact or spayed concurrently with mass
effect of multiple criteria on OS, and hazard ratios were calcu- removal. Tumor diameter was less than 2 cm in 51 cases
lated using the Mantel-Haenszel method. For continuous vari- (52.6%), between 2 and 3 cm in 18 cases (18.4%), greater than
ables, categorization was based on median values or tertiles 3 cm in 19 cases (19.6%), and unknown in 9 cases (9.2%). On
unless otherwise stated. OS was defined as the period between presentation to the referring clinic, 56 cats (57.7%) were
the date of biopsy/excision and the date of death or last docu- reported to have a single mass, 40 (40.8%) had multiple
mented follow-up. Cases were grouped into 5 distinct out- masses, and the number of masses was unreported in 1. A total
comes: (1) alive, (2) lost to follow-up, (3) tumor-related of 43 cases (43.9%) were classified as WHO stage 1, 15
death/euthanasia, (4) non–tumor-related death/euthanasia, and (15.3%) as stage 2, and 32 (33%) as stage 3, with 7 (7.2%)
(5) death/euthanasia of unverified cause (but suspected to be unknown (survey information incomplete).
tumor related). For statistical analysis of OS, outcomes 1, 2, Results of Kaplan-Meier survival analysis of survey data
and 4 were considered censored events. In all cases, a P value and histologic features are listed in Tables 3, 4, and 5. Although
equal to or less than .05 was considered significant. tumor diameter was not a significant factor overall, those
greater than 3 cm in diameter were associated with reduced
OS (P ¼ .046) compared with tumors less than 2 cm in dia-
Results meter. More advanced WHO stage and the presence of lymph
Female cats in this study ranged in age from 3.5 to 20 years old node metastases also showed statistically significant negative
(median, 11 years; mean [SD], 11.4 [3.5] years). Breeds correlation with OS (both P ¼ .01). Both WHO histologic sub-
included 65 domestic short hair (66.3%), 14 domestic long hair type (P ¼ .005) and the presence of lymphovascular invasion
(14.4%), 5 domestic medium hair (5.1%), 6 Siamese (and (P < .001) corresponded with OS. A weak association was
crosses) (6.2%), 2 Maine Coon (and crosses) (2%), 1 Korat found between tumors with >75% tubule formation and those
cross (1%), 1 Manx (1%), 1 Birman (1%), 1 Chinchilla (1%), with less than 10% tubule formation (P ¼ .037) and OS, the
Mills et al 243

Table 2. Novel Grading System for Evaluation of Invasive Mammary Carcinoma in Female Cats.

Histologic Featurea Score

Lymphovascular invasion
Absent 0
Present 1
Nuclear formb
5% abnormal 0
>5% abnormal 1
Mitotic countc
62 0
>62 1

Total Score Grade Median Survival, mo Survival at 18 mo, %

0 I (low-grade carcinoma) 31 82
1 II (intermediate-grade carcinoma) 14 37
2–3 III (high-grade carcinoma) 8 18
a
Each feature is evaluated and scores are assigned and summed. Absence of lymphovascular invasion, abnormal nuclear form 5%, and a mitotic count 62
correspond to grade I (total score ¼ 0). The presence of any one of lymphovascular invasion, abnormal nuclear form >5%, or a mitotic count >62 indicates grade II
(total score ¼ 1). If any 2 or all 3 features are present, grade III is assigned (total score ¼ 2–3).
b
Abnormal nuclear form includes any deviation from smooth nuclear contour or round/oval nuclear shape such as clefting, angularity, corrugation, or ameboid
morphology assessed at high power (40–60 objective) in the least differentiated and/or most invasive portions of the tumor (Figs. 1–4). The number of nuclei
exhibiting the abnormal nuclear form is estimated and expressed as a percentage of the total number of nuclei within any given field.
c
Cumulative number of mitoses in 10 consecutive fields in the most mitotically active area with a microscope field diameter of 0.53 mm (40 objective).

Table 3. Kaplan-Meier Analysis of Selected Clinicopathologic Criteria With Respect to Overall Survival in Female Cats With Mammary
Carcinoma.

na (Median Survival in mo) P Value Hazard Ratio 95% CI

Age (median, 11 y) .9 1 0.66–1.6

(i)  11 y 50 (15) — — —
(ii) > 11 y 47 (14) — — —
Reproductive status .2 1.5 0.8–2.9
(i) Intact 15 (11) — — —
(ii) Spayed 82 (15) — — —
Tumor diameter .09
(i) <2 cm 51 (16) .17b 0.65b 0.35–1.2
(ii) 2–3 cm 18 (14) .49c 0.78c 0.38–1.6
(iii) >3 cm 19 (11) .046d 0.50d 0.25–1.0
Lymph node metastases .01 0.39 0.19–0.8
(i) No 66 (16) — — —
(ii) Yes 17 (9) — — —
WHO stage .01
(i) 1 43 (18) .19b 0.63b 0.31–1.3
(ii) 2 15 (15) .20c 0.66c 0.35–1.3
(iii) 3 32 (10) .004d 0.43d 0.25–0.7
CI, confidence interval; WHO, World Health Organization; —, .
a
Number of cats for which survey data were available.
b
i vs ii.
c
ii vs iii.
d
i vs iii.

latter conferring a poorer prognosis. The median survival of evaluated within the neoplastic population were significantly
cats with solid or cribriform carcinomas (10 and 8 months, and inversely related to OS, including abnormal nucleolar mor-
respectively) was less than half that of cats with the tubulopa- phology (P ¼ .013), increased anisokaryosis (P ¼ .013),
pillary subtype (21 months). Percentage of necrosis, the pres- increased size (P ¼ .038), abnormal nuclear form (P < .001),
ence or nature of any inflammation, the stromal response, increased nuclear pleomorphism score (P ¼ .006), and
and the percentage of squamous differentiation were not statis- increased mitotic count (P ¼ .021). Visible nucleoli were
tically significant with respect to OS. In contrast, with the absent in only 2 tumors (1.9%); nuclear size was increased rela-
exception of chromatin vesiculation, all nuclear characteristics tive to normal in 104 tumors (96.3%). The mean mitotic count
244 Veterinary Pathology 52(2)

Table 4. Kaplan-Meier Analysis of Low-Power Histologic Criteria With Respect to Overall Survival in Female Cats With Mammary Carcinoma.

na (Median Survival in mo) P Value Hazard Ratio 95% CI

Tumor subtype .005

(i) Tubulopapillary 61 (21) <.001b 0.41b 0.24–0.68
(ii) Solid 36 (10) .95c .98c 0.42–2.2
(iii) Cribriform 10 (8) .043d .32d 0.10–0.97
(iv) Squamous cell carcinoma 1 (—) — — —
Tubule formatione .1
(i) <10% 37 (13) .45b 1.2b 0.74–1.9
(ii) 10%–75% 54 (14) .08c 1.7c 0.93–3.2
(iii) >75% 17 (29) .037d 2.0d 1.0–3.7
Necrosis .1 0.69 0.44–1.1
(i) 25% 56 (18) — — —
(ii) >25% 52 (12) — — —
Inflammation .22
(i) None present or mild 34 (18) .23b 0.74b 0.45–1.2
(ii) Lymphoplasmacytic 59 (15) .33c 0.71c 0.36–1.4
(iii) Neutrophilic or pleocellular 15 (9) .15d 0.57d 0.27–1.2
Lymphovascular invasion <.001 0.38 0.23–0.62
(i) Absent 68 (18) — — —
(ii) Present 40 (8) — — —
Stromal response .16
(i) Normal (mild-moderate) 71 (14) .15b 1.4b 0.88–2.3
(ii) Marked 29 (14) .61c 1.3c 0.48–3.5
(iii) Intratumoral 8 (31) .13d 1.8d 0.84–3.8
Squamous differentiation .2 0.74 0.46–1.2
(i) 5% 67 (15) — — —
(ii) >5% 41 (12) — — —

CI, confidence interval; —, .

a
Number of tumors.
b
i vs ii.
c
ii vs iii.
d
i vs iii.
e
Evaluated according to the Elston and Ellis grading system.

was 67 mitotic figures per 10 high-power fields, with a median (n ¼ 61) and 13 months (n ¼ 18) for cats with grade II and III
value of 62. When divided into tertiles, categories were 0 to 50, carcinomas, respectively. REE grading resulted in 32 carcino-
51 to 70, and >71 mitotic figures per 10 high-power fields. In mas (29.6%) being classified as grade I (median survival 29
contrast, when mitotic counts were grouped according to the months). This was 2.5-fold longer OS than grade II carcinomas
EE grading system, 1 tumor (1.0%) was given a score of 1, 4 (median survival 12 months; n ¼ 63) and 5.8-fold longer
(3.7%) a score of 2, and 103 (95.4%) a score of 3. OS than grade III carcinomas (median survival 5 months;
Only lymphovascular invasion, mitotic count, and nuclear n ¼ 13). When individual grades were analyzed separately, a
form could be considered independent prognostic factors in the statistically significant difference was not identified between
current study (P ¼ .008, .004, and <.001, respectively) accord- grades II and III in the MMEE or the REE grading system.
ing to Cox proportional hazards analysis (Suppl. Table S1). When tumors were graded histologically using the novel sys-
Overall, EE grading was not associated with OS (Fig. 5). A sta- tem, a statistically significant difference was present between
tistically significant difference was found between grades II and grades I and II (P ¼ .0012), grades II and III (P ¼ .024), and
III (P ¼ .026), with a median survival of 17 months (n ¼ 63) and grades I and III (P < .0001). Cats with grade III (high-grade)
13 months (n ¼ 43), respectively. Only 2 tumors (1.9%) received carcinomas had only one-fourth (25.8%) the survival time of
a nuclear pleomorphism score of 1, and only 5 (4.6%) received a cats with grade I (low-grade) carcinomas (median survival
mitotic count score of either 1 or 2 (data not shown). 8 months [n ¼ 48] and 31 months [n ¼ 22], respectively) and
A strong association was found between the MMEE grading less than two-thirds (57%) the median survival time of cats
and OS (P < .001; Fig. 6), REE grading and OS (P < .001; Fig. with grade II carcinomas (median survival 14 months; n ¼ 38).
7), and novel grading and OS (P < .001; Fig. 8). The MMEE
grading system classified 29 carcinomas (26.9%) as grade I
(median survival of 27 months), compared with only 2 grade Discussion
I carcinomas (1.9%) using the original EE grading system To our knowledge, this is the largest histologic analysis of
(median survival 19 months). Median survival was 14 months feline mammary carcinoma to date, aimed at evaluating and
Mills et al 245

Table 5. Kaplan-Meier Analysis of High-Power Histologic Criteria With Respect to Overall Survival in Female Cats With Mammary Carcinoma.

na (Median Survival in mo) P Value Hazard Ratio 95% CI

Chromatin vesiculation .96

(i) Absent or mild 71 (13) .99b 1.0b 0.62–1.6
(ii) Moderate 34 (15) .71c 0.78c 0.20–3.0
(iii) Marked 3 (18) .84d 0.88d 0.26–3.0
Nucleoli .013
(i) Indistinct 2 (34) .58b 0.65b 0.14–3.0
(ii) Single and small 96 (15) .004c 0.2c 0.07–0.59
(iii) Abnormal and/or multiple 10 (4) .43d 0.52d 0.1–2.7
Anisokaryosis .013
(i) <2-fold variation 69 (18) .048b 0.47b 0.28–0.8
(ii) 2- to 3-fold variation 35 (9) .8c 1.1c 0.42–3.1
(iii) >3-fold variation 4 (14) .18d 0.38d 0.1–1.5
Nuclear size .038
(i) Normal 4 (35) .027b 0.38b 0.16–0.89
(ii) 2-fold larger than normal 89 (14) .35c 0.73c 0.38–1.4
(iii) >2-fold larger than normal 15 (16) .002d 0.14d 0.04–0.48
Nuclear form <.001
(i) 5% abnormal 64 (21) <.001b 0.35b 0.2–0.61
(ii) 5%–25% abnormal 36 (12) .85c 1.1c 0.48–2.4
(iii) >25% abnormal 8 (12) .12d 0.45d 0.16–1.2
Nuclear pleomorphism scoree .006
(i) 1 2 (34) .68b 0.71b 0.14–3.7
(ii) 2 83 (16) .002c 0.38c 0.2–0.69
(iii) 3 23 (11) .21d 0.43d 0.12–1.6
Mitotic countf (median, 62) .021 0.59 0.37–0.92
(i) 62 54 (18) — — —
(ii) >62 54 (9) — — —
CI, confidence interval; —, .
a
Number of tumors.
b
i vs ii.
c
ii vs iii.
d
i vs iii.
e
Evaluated according to the Elston and Ellis grading system.
f
Cumulative number of mitoses in 10 consecutive fields in the most mitotically active area with a microscope field diameter of 0.53 mm (40 objective).

Figure 6. Kaplan-Meier curve depicting mitotic-modified Elston and

Figure 5. Kaplan-Meier curve depicting Elston and Ellis grade (Table Ellis grade (Table 1) vs overall survival in female cats with invasive
1) vs overall survival in female cats with invasive mammary carcinoma. mammary carcinoma. Median survival was 27 months, 14 months, and
Median survival was 19 months, 17 months, and 13 months for grade I, 13 months for grade I, II, and III tumors, respectively. Hashmarks indi-
II, and III tumors, respectively. Hashmarks indicate censored events. cate censored events.

improving current grading methodologies. Due to the current malignant mammary carcinomas in diagnostic practice is not
lack of a globally accepted, reliable, species-specific grading typically revised as a result of histologic assessment. It was evi-
system, the poor prognosis generally conferred upon cats with dent in this and previous studies, however, that survival times
246 Veterinary Pathology 52(2)

reduced survival times, tumor size alone appears to have little

or no independent prognostic value when smaller than 3 cm in
diameter.25,36
The modified WHO clinical stage provides a useful prog-
nostic indicator in cats with advanced clinical disease.15 Stage
3 cats had significantly reduced OS compared with stage 1 cats
in this study; a statistical association between WHO stage and
OS has also been noted elsewhere.34 Unfortunately, WHO sta-
ging relies heavily on tumor diameter and may therefore be
inadequate for cats with more localized disease at the time of
diagnosis or where regional metastases are not detected (stages
I and II).
Application of the EE grading system to our study popula-
Figure 7. Kaplan-Meier curve depicting revised Elston and Ellis grade tion resulted in inferior discrimination between tumors and
(Table 1) vs overall survival in female cats with invasive mammary car- lacked overall association with OS. Tumors were heavily
cinoma. Median survival was 29 months, 12 months, and 5 months for skewed toward grade II or III (58.3% and 39.8% of carcino-
grade I, II, and III tumors, respectively. Hashmarks indicate censored mas, respectively), resulting in underrepresentation of grade I
events. (well-differentiated) carcinomas (1.9% of evaluated tumors).
The asymmetry of this classification stemmed directly from
nuclear pleomorphism and mitotic count results, both of
which were heavily weighted toward higher scores (data not
shown).
These results contrast with previous studies, where the EE
grading system has shown significant prognostic utility in
intact queens.4,19,34 It was noted, however, in the prospective
study by Castagnaro et al4 that a grade II designation lacked
prognostic value, a category that comprised a majority of the
tumors they evaluated. Furthermore, survival analysis was not
conducted in that study, limiting the conclusions that may be
drawn. In the report by Millanta et al,19 significant overlap is
evident on the Kaplan-Meier curves depicting EE grade vs
OS, and it is unclear to which group (grade) comparisons the
statistical significance is attributable. The retrospective inves-
Figure 8. Kaplan-Meier curve depicting novel grade (Table 2) vs over- tigation by Seixas et al34 is most comparable to the present
all survival in female cats with invasive mammary carcinoma. Median
study in both scope and objective. Similar to our findings, only
survival was 31 months, 14 months, and 8 months for grade I, II, and
III tumors, respectively. Hashmarks indicate censored events. 5.4% of tumors they evaluated were given a grade I designation
using the EE grading system, perhaps further underscoring the
need for a more discriminating grading method. Other possible
in affected cats are variable, with postsurgical survival extend- explanations for the discrepant results found in this study
ing for years rather than months in many cases.19,29,36 Findings include the inherent subjectivity associated with tumor grad-
of this study support the use of modified EE grading systems or ing,23 differences in population-specific factors (ie, spayed vs
a novel grading system to improve the prognostic value of his- intact, geographic location), differences attributable to study
tologic grading for feline mammary carcinoma. type and case definition, or an inherently deficient grading
Tumor size (or diameter) is easily estimated at presentation system.
to a primary practice and is a commonly used diagnostic indi- To address the latter possibility, numerous morphological
cator due to its reported correlation with prognosis in feline criteria were evaluated in the present study and analyzed with
mammary carcinoma.14,16,39 Our study demonstrated only a respect to OS. Tumor subtypes were included, despite their
weak statistical association between tumor diameter and OS characterization as descriptive rather than prognostic.20 Indeed,
and only for tumors greater than 3 cm in diameter. This may a significant association between tumor subtype and OS was
be due to insufficient numbers as well as the inherent subjectiv- found here and by Seixas et al34 but not by Millanta et al19
ity involved in measurement of tumor diameter (calipers vs pal- or Castagnaro et al.4 This discrepancy may reflect the inherent
pation, in vivo vs ex vivo, etc). Additional shortcomings and subjectivity of histologic evaluation, the morphological hetero-
possible sources of error encountered when using tumor size geneity of feline mammary carcinoma, or variable interpreta-
(or volume) as a prognostic factor are described by Matos tion of the category descriptions.20
et al.18 Recent reports support findings here, indicating that Both the mitotic count and the presence of lymphovascular
while larger tumors (>3 cm diameter) are associated with invasion demonstrated a strong correlation with OS, similar to
Mills et al 247

findings in previous studies.29,34 Mitotic index and histologic include the additional time required for evaluation of a fourth
staging based on lymphovascular invasion were both found criterion and familiarization with nuclear form scoring.
to be independent prognostic factors by Preziosi et al.29 The A novel grading system was also developed, based solely on
prognostic significance of invasion was also recognized by those criteria found to be independent prognostic factors: lym-
Mandelli et al,17 who developed a histologic grading system for phovascular invasion, mitotic count, and nuclear form. Only
feline mammary gland tumors based on this feature alone. 2 subcategories were established for mitotic count and nuclear
In this study, nucleolar morphology, anisokaryosis, nuclear form to simplify the grading system. When applied to the study
size, nuclear form, and nuclear pleomorphism score all showed population, a statistically significant difference was found
a significant correlation with OS in univariate analysis. Upon between all grades in terms of OS. Although fewer were iden-
multivariable analysis, however, nuclear form was the only tified relative to MMEE and REE grading, grade I (low grade)
aspect of nuclear morphology shown here to be an independent carcinomas were well represented. The presence of only 2 cate-
prognostic factor in feline mammary carcinoma. Nuclear form gories associated with each criterion greatly simplifies histolo-
was previously evaluated using nuclear morphometry software. gic scoring using this system, increasing the potential utility for
DeVico and Maiolino6 concluded that both the standard devia- the diagnostic pathologist. In addition, cats with grade I (low-
tion and coefficient of variation of nuclear form were reliable grade) carcinomas were again more adequately represented by
prognostic factors in cats with mammary carcinoma. In a more the novel grading system compared with the EE grading
recent study, however, Simeonov and Simeonova35 found no system.
statistical correlation between nuclear morphometric para- A potential limitation associated with use of lymphovascu-
meters and OS upon analysis of cytological samples from feline lar invasion in the REE and novel grading systems is the incon-
mammary carcinomas. Further investigation is warranted given sistent presence between histologic sections of the same tumor.
the results of the present study. While it is true that the extent to which identifying lymphovas-
In light of the multivariate analysis results together with the cular invasion depends on which portion(s) or serial section(s)
apparent shortcomings of EE grading when applied to the study of the tumor are evaluated, the impact this has on its value as a
population, 3 new grading systems were developed in an prognostic indicator is unknown. There is suboptimal sensitiv-
attempt to improve histologic grading of feline mammary car- ity associated with many histologic criteria evaluated on a 2-
cinoma. First, as has been recently described for grading canine dimensional section of a 3-dimensional, heterogeneous tumor.
mammary carcinomas,27 modifications of the EE grading sys- Despite these potential shortcomings, vascular invasion has
tem were proposed. The MMEE grading system features been included in histologic grading systems for other tumors,
increased range subcategories within the mitotic count including squamous cell carcinoma of the tongue in dogs,3 as
category to better accommodate the high median and broad well as for prognostication in feline primary lung carcinomas.10
range of mitotic counts encountered within our study popula- In contrast to dogs, where the protective effect of early
tion. Application of the MMEE grading system greatly ovariohysterectomy on the development of mammary carci-
improved detection of grade I (well-differentiated) carcinomas noma is well established, less is known about the impact
compared with the EE grading system, both in terms of the of reproductive hormones on feline mammary carcinoma.
number of tumors recognized and median survival. The REE Overley et al26 demonstrated a clear protective effect of ovar-
grading system represents a more extensive modification of iohysterectomy, whereby those cats having undergone the
the EE grading system to further improve tumor discrimina- procedure prior to 1 year of age showed an 86% reduction
tion. Nuclear pleomorphism, a significant source of skewness in risk of mammary carcinoma relative to intact cats. Unfor-
within the data and long recognized as the most subjective cri- tunately, scant information is available regarding possible
terion in the EE system,7 was removed and replaced by effect(s) of spay status on tumor characteristics, grading, and
nuclear form. Although still admittedly subjective, this allows OS in cats with mammary carcinoma. No difference was
the diagnostic pathologist to concentrate on a single aspect of found between spayed and intact cats with regard to OS in the
nuclear morphology irrespective of concurrent nuclear fea- present study, although median survival was 66% higher in
tures. Lymphovascular invasion was also added, recognizing the former group. This may simply be due to a lack of statis-
its prognostic significance. REE grading again resulted in tical power, as only 15% (n ¼ 16) of carcinomas were from
superior recognition of grade I (well-differentiated) carcino- intact animals. In dogs with mammary carcinoma, spay status
mas compared with EE grading. In addition, grade III (poorly has been shown to be significantly associated with sur-
differentiated) carcinomas were more adequately represented vival.5,27 Recently, Hughes and Dobson13 called attention to
in terms of median survival. It should be noted, however, that the need for larger studies to investigate the effect of spay sta-
neither the MMEE nor the REE grading system was able to tus on prognostic marker expression in feline mammary carci-
show significant differences between grades II and III. In the nomas. It is currently unknown to what extent the effects of
latter case, this may be attributable to the low number of grade endogenous estrogen and/or progesterone exposure may man-
III carcinomas. ifest histologically and/or immunohistologically in feline
In practical terms, grading tumors with the MMEE grading mammary carcinoma. If present, this may influence diagnos-
system is virtually identical to using the original EE grading tic evaluation, including any attempts at histologic grading.
system. Potential drawbacks of the REE grading system Further investigation is required in this area.
248 Veterinary Pathology 52(2)

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