Oncology–Original Article
Prognostic Value of Histologic Grading
for Feline Mammary Carcinoma:
A Retrospective Survival Analysis
Veterinary Pathology
2015, Vol. 52(2) 238-249
ª The Author(s) 2014
Reprints and permission:
sagepub.com/journalsPermissions.nav
DOI: 10.1177/0300985814543198
vet.sagepub.com
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 primary veterinary practice, accounting for 17% of neoplasms in
female cats.15 Reported malignancy rates generally range
between 80% and 90%,9,15 with fibroadenoma, fibroadenomatous hyperplasia, or duct ectasia being the most commonly
encountered benign, dysplastic, or nonneoplastic masses,
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
aggressive, survival times can vary significantly.19,29,36 Prognosis in affected cats has been shown previously to be influenced
by tumor diameter,14,16,36,38,39 World Health Organization
(WHO) stage and modified WHO stage,14,34 extent of surgery,16,25 and histologic grade.4,34 The Elston and Ellis (EE) histologic grading system (also known as the Nottingham
Grading System) has been widely adopted by veterinary
investigators for the grading of feline mammary carcinoma.2,4,19,28,32–34,40 This system represents the ‘‘gold standard’’ in assessment of invasive human breast cancer,
whereby distinct histopathologic features (ie, percentage
tubule formation, nuclear pleomorphism, and mitotic count)
are scored and added together to produce a grade, which
then correlates with degree of malignancy and prognosis.8,30,31 Use of this grading system by veterinary pathologists is based primarily on evidence suggesting that feline
mammary carcinoma represents a suitable model for human
*
Indicates equal contribution
Western College of Veterinary Medicine, University of Saskatchewan,
Saskatoon, SK, Canada
2
Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
3
College of Veterinary Medicine and Biomedical Sciences, Colorado State
University, Fort Collins, CO, USA
1
Supplemental material for this article is available on the Veterinary Pathology
website at http://vet.sagepub.com/supplemental.
Corresponding Author:
S. Mills, Western College of Veterinary Medicine, University of Saskatchewan,
52 Campus Drive, Saskatoon, SK, Canada.
Email: [email protected]
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
Mills et al
239
breast cancer, particularly the more aggressive estrogennegative carcinomas.2,22,39,41
Castagnaro et al4 found the EE grading system to have
good predictive value for well-differentiated (grade I) and
poorly differentiated (grade III) carcinomas. The same grading method was found to have high predictive value in
queens with invasive carcinomas by Millanta et al.19 More
recently, Seixas et al34 found histologic grade determined
using the EE grading system to be an independent prognostic factor for middle-aged to elderly queens with mammary
carcinoma. These studies present compelling preliminary
evidence in support of histologic grading using the EE grading system as a predictor of overall survival (OS) time in
unspayed female cats.
Matos et al18 recently called attention to the need for a
standardized grading system for feline mammary gland carcinomas, where early detection, reliable histologic characterization, and aggressive treatment may have a significant
influence on survival times. The aim of the current study
was to (1) determine whether histologic grade using the
EE grading system is a reliable prognostic indicator for a
predominantly spayed, North American population of
female cats with mammary carcinoma and (2) determine
whether modification of the EE grading system and/or
development of a novel grading system would improve the
prognostic value of routine histologic examination of feline
mammary carcinoma.
Materials and Methods
Case Origin and Data Collection
Cases were drawn from 834 surgical biopsy specimens of
mammary tumors originating from 789 domestic cats and submitted to either Veterinary Diagnostic Services (later Prairie
Diagnostic Services, Inc) at the University of Saskatchewan
between 1989 and 2011 or to the Veterinary Diagnostic
Laboratories at Colorado State University between 2005 and
2010. Use of animal tissues in this study was approved by the
University Committee on Animal Care and Supply at the University of Saskatchewan. A total of 329 surveys were distributed to referring clinics to obtain follow-up information based
on the original medical case file. Partial or complete information on 225 cases comprising 255 excisional biopsy specimens (tumors) was obtained. The survey collected the
patient signalment; size, location, appearance, and duration
of mammary tumor(s); presence and location of lymph node
and/or distant metastasis at initial diagnosis; presence and
timing of local/regional recurrence or metastases; and the timing and nature of the eventual outcome. All cases were staged
according to the modified WHO staging system.15 Briefly,
stage I and stage II included cats with primary tumors less
than 2 cm and 2 to 3 cm in diameter, respectively, with no evidence of regional or distant metastases. Stage III included cats
with primary tumors less than 2 cm or 2 to 3 cm in diameter
with evidence of regional metastases or cats with tumors
greater than 3 cm in diameter with or without evidence of
regional metastases. Stage IV included any cat with evidence
of distant metastases regardless of tumor size or regional
metastases.
Inclusion/Exclusion Criteria
This study included only primary mammary tumors from
female cats (spayed or intact) with complete survey data, a specimen of adequate quality, a confirmed histologic diagnosis of
invasive mammary carcinoma, and a WHO clinical stage of I,
II, or III. Except in cases where death or euthanasia occurred
and was determined or suspected to be tumor related, at least
1 year of follow-up information was required for inclusion.
Any cats receiving treatment beyond surgical excision of the
tumor (ie, drug or radiation therapy) were excluded. Noninfiltrating (in situ) carcinomas were excluded. A tumor was considered primary when there was no prior history of mammary
neoplasia indicated in the pathology report or survey data.
Every effort was made to confirm mammary origin. In cases
that initially presented with multiple mammary masses, all submitted tumors were evaluated independently. A total of 97 cats
fulfilled the case definition, encompassing 108 tumors. Of
those, 88 (81%) were single tumor submissions, while 20
(19%) were multiple tumor submissions from the same animal.
Processing of Tissues
Archived, paraffin-embedded tissues were retrieved, sectioned
(4 mm), and stained with hematoxylin and eosin (HE). When it
was necessary to confirm intravascular invasion, vascular
endothelial cells were highlighted by immunohistochemical
staining for Von Willebrand factor carried out on paraffinembedded sections using a commercial staining platform
(Benchmark staining platform; Ventana Medical Systems,
Tucson, AZ) and a streptavidin-biotin detection system (BMK
iVIEW DAB Paraffin detection kit; Ventana Medical Systems). The primary antibody (polyclonal rabbit anti–human
Von Willebrand factor; Dako Canada, Inc, Mississauga, ON,
Canada) was applied for 32 minutes at a dilution of 1:1000.
Histologic Assessment Criteria
Histologic slides were reviewed independently by 2 authors
(S.W.M. and K.M.M.), with direct assistance from two ACVP
board-certified veterinary pathologists (J.L.D. and E.S.).
Assessors were blinded to the clinicopathologic (survey) data
and clinical outcome; all discordant results were reviewed by
S.W.M. and K.M.M. with a multiheaded microscope to reach
consensus. When more than 1 section was available for examination, only the one containing the largest portion of the tumor
was selected for evaluation. The only exception was assessment of lymphovascular invasion, where all available sections
were screened. All tumors were initially classified according to
the WHO subtypes described for malignant feline mammary
carcinomas.20 The current study included tubulopapillary,
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
240
Veterinary Pathology 52(2)
solid, cribriform, and squamous cell carcinomas, with most
demonstrating more than 1 growth pattern. In these instances,
the pattern comprising the largest relative portion of the tumor
determined the subtype.
Tumor characteristics evaluated included percentage tubule
formation, necrosis, squamous differentiation, inflammation,
lymphovascular invasion, stromal response, chromatin vesiculation, nucleolar morphology, anisokaryosis, nuclear size,
nuclear form, nuclear pleomorphism, and mitotic count. Percentage tubule formation was evaluated according to Elston
and Ellis.8 Briefly, the proportion of neoplastic cells within a
given section exhibiting discrete tubule formation relative to
the total tumor mass was expressed semi-quantitatively as a
percentage. Necrosis and squamous differentiation were scored
in similar fashion. Changes consistent with squamous differentiation included at least 2 of the following: decreased N:C ratio,
increased cytoplasmic eosinophilia, increased cell size, and
increased angularity of cell margin. Inflammation was classified subjectively as follows: (1) absent or very mild, (2) predominantly lymphoplasmacytic, or (3) neutrophilic or
pleocellular. A tumor was positive for lymphovascular invasion if neoplastic emboli were clearly seen within a continuous,
endothelium-lined vessel lumen. In cases where the assessment of lymphovascular invasion was equivocal or consensus
could not be reached, additional sections were stained for Von
Willebrand factor to highlight vascular endothelium and a
definitive classification made. Stromal response encompassed
desmoplasia, myofibroblastic/myoepithelial hyperplasia, or a
combination of both; it was often not possible to definitively
discriminate between these processes in HE-stained tissue
sections. The stromal response was graded as normal (mildmoderate) or marked when limited primarily to the periphery
of the tumor, or intratumoral when it dissected through the
tumor in a trabecular fashion. Assessment of neoplastic cell
nuclei was limited to the least differentiated and/or most invasive portions of the tumor in areas lacking appreciable squamous
differentiation to reduce bias. Normal nuclear morphology was
defined as that which may be found in normal mammary tubular
epithelium. Subcategories for chromatin vesiculation, nucleolar
morphology, anisokaryosis, nuclear size, and nuclear form were
developed and assessed subjectively based on the range of
morphology observed in all specimens. Chromatin vesiculation
evaluated the predominance of euchromatin within the nuclear
envelope as evidenced by areas of pallor or clear space. Categories included (1) none or mildly vesiculated, (2) moderately
vesiculated, or (3) markedly vesiculated. Nucleoli were characterized as (1) indistinct, (2) small and prominent, or (3) large,
multiple, or abnormally shaped. Anisokaryosis and nuclear size
were expressed relative to adjacent tumor cells and nuclei in normal mammary epithelium, respectively. The nuclear form
assessment derived from a high-power (40–60 objective), subjective evaluation of nuclear shape independent of other nuclear
features or artifactual changes (Figs. 1–4, Suppl. Figs. 1–6).
Deviations from a smooth nuclear contour and round or oval
shape such as corrugation, angularity, clefting (indentation), or
overtly ameboid shape were considered abnormal. Several fields
were evaluated in the least differentiated and/or most invasive
portion of the tumor, and the number of nuclei exhibiting abnormal form was estimated relative to the total number of nuclei
within a given field and expressed as a percentage. Subcategories (5%, 6%–25%, or >25% abnormal) were then assigned.
Mitotic figures were evaluated according to Elston and Ellis.8
Briefly, mitotic figures were counted in 10 consecutive fields
at the periphery of the tumor in the areas of highest proliferative
activity. Microscope field diameter was maintained at 0.53 mm
for all observations to standardize counts.1 Care was taken
to exclude apoptotic, pyknotic, or otherwise hyperchromatic
nuclei, only including those that had definitively entered prophase or were in metaphase, anaphase, or telophase. In certain
cases where tumor heterogeneity and high mitotic rate caused
mitotic count to differ by more than 20% between 2 observers,
they were repeated independently until acceptable concordance
was attained. A correlation coefficient (Pearson’s R value) of
0.954 was obtained overall.
Histologic Grading
Initially, tumors were graded according to the EE grading system described for human breast cancer (Table 1).8 In this system, carcinomas are scored according to 3 criteria: percentage
tubule formation, degree of nuclear pleomorphism, and mitotic
count. Briefly, the degree of tubule formation is assessed subjectively on low power and expressed as a percentage. When
greater than 75% of the tumor parenchyma exhibits tubule formation, 1 point is assigned. Tubule formation between 10% and
75% is given a score of 2 points, and 3 points are assigned when
<10% of the tumor parenchyma displays tubule formation.
Nuclear pleomorphism scoring is conducted at high power
(40) in the least differentiated and/or most invasive portion
of the tumor, typically along the periphery. Tumor cell nuclei
that display uniform chromatin distribution with only mild variation in size, shape, and contour relative to normal nuclei are
assigned 1 point. Enlarged nuclei with more vesicular chromatin and moderate variation in size and shape are given 2 points.
Finally, 3 points are assigned when tumor cell nuclei exhibit
marked vesiculation as well as marked variation in size and
shape. Nucleoli are very often present in the latter 2 cases; multiple nucleoli or nucleolar atypia favor category 3.
Mitotic counts were grouped as follows: a total of 0 to 8
mitotic figures in 10 high-power fields (40) was given 1
point, 9 to 16 was given 2 points, and >17 was given 3 points.
The score for each category was summed, and the total score
for each tumor corresponded to a predetermined grade (I, II,
or III), indicating well-differentiated, moderately differentiated, or poorly differentiated carcinomas, respectively.
Three new grading systems were designed, applied to our
data, and analyzed with respect to OS. First, in the mitoticmodified Elston and Ellis (MMEE) grading system (Table 1),
range subcategories within the mitotic count category of the
EE grading system were modified to better accommodate the
wide range and high magnitude of mitotic counts observed
within the tumors we evaluated. Cutoff values between mitotic
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
Mills et al
241
Figures 1–4. Mammary gland; cat, mammary carcinoma. Hematoxylin and eosin. Figure 1. Example of low nuclear form score (5% abnormal). 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 boundaries from our results. Next, with the revised Elston and Ellis
(REE) grading system (Table 1), the EE grading system was
modified further to include nuclear form scoring and lymphovascular invasion. Nuclear pleomorphism scoring was
removed, and an additional point was added to the grading
chart to accommodate the addition of lymphovascular invasion.
Scoring and grading with the MMEE and REE grading systems
was otherwise achieved in the same manner as described above
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
prognostic factors (lymphovascular invasion, nuclear form, and
mitotic count) using coefficients from the model and divided
into grades. Mitotic count subcategories were derived directly
from the median mitotic count observed in our results. Using
the novel grading system, the absence of lymphovascular
invasion together with less than or equal to 5% abnormal
nuclear form and a mitotic count less than or equal to 62 in
10 high-power fields corresponded to grade I (low-grade carcinoma). The presence of any one of lymphovascular invasion,
greater than 5% abnormal nuclear form, or a cumulative mitotic
count greater than 62 yielded grade II (intermediate-grade carcinoma). Finally, if any 2 or all 3 of the aforementioned features were present, grade III (high-grade carcinoma) was
assigned.
Statistical Analysis
Analyses were conducted using Prism (GraphPad Software,
La Jolla, CA) and Stata (StataCorp LP, College Station TX)
statistical software packages. Normality was assessed with the
D’Agostino and Pearson normality test. To analyze the relationship between individual survey or histologic criteria/
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
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
EE, MMEE, REE
EE, MMEE
EE, MMEE, REE
REE only
REE only
Histologic Feature
Tubule formation
Comprises a majority of the tumor (>75%)
Present to a moderate degree (10%–75%)
Little or none present (<10%)
Nuclear pleomorphism
Small, regular, uniform nuclei
Moderately increased size, vesiculation, and variability
Vesicular chromatin with marked variation in size and shape
Mitotic counta
EE
MMEE, REE
0–8
0–50
9–16
51–70
17
71
Lymphovascular invasion
Absent
Present
Nuclear formb
5% abnormal
6%–25% abnormal
>25% abnormal
Point Total
Grade
Comment
3-5
6-7
8-9 or 8-10 (REE)
I
II
III
Well differentiated
Moderately differentiated
Poorly differentiated
Score
1
2
3
1
2
3
1
2
3
0
1
1
2
3
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).
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.
b
grading and OS, Kaplan-Meier curves were generated and
compared using log rank or Gehan-Breslow-Wilcoxon tests.
A Cox proportional hazards model was used to evaluate the
effect of multiple criteria on OS, and hazard ratios were calculated using the Mantel-Haenszel method. For continuous variables, categorization was based on median values or tertiles
unless otherwise stated. OS was defined as the period between
the date of biopsy/excision and the date of death or last documented follow-up. Cases were grouped into 5 distinct outcomes: (1) alive, (2) lost to follow-up, (3) tumor-related
death/euthanasia, (4) non–tumor-related death/euthanasia, and
(5) death/euthanasia of unverified cause (but suspected to be
tumor related). For statistical analysis of OS, outcomes 1, 2,
and 4 were considered censored events. In all cases, a P value
equal to or less than .05 was considered significant.
Results
Female cats in this study ranged in age from 3.5 to 20 years old
(median, 11 years; mean [SD], 11.4 [3.5] years). Breeds
included 65 domestic short hair (66.3%), 14 domestic long hair
(14.4%), 5 domestic medium hair (5.1%), 6 Siamese (and
crosses) (6.2%), 2 Maine Coon (and crosses) (2%), 1 Korat
cross (1%), 1 Manx (1%), 1 Birman (1%), 1 Chinchilla (1%),
and 1 Devon Rex (1%). Ovariectomy or ovariohysterectomy
(spaying) had been performed previously in 82 cats (84.7%),
while 15 (15.3%) were intact or spayed concurrently with mass
removal. Tumor diameter was less than 2 cm in 51 cases
(52.6%), between 2 and 3 cm in 18 cases (18.4%), greater than
3 cm in 19 cases (19.6%), and unknown in 9 cases (9.2%). On
presentation to the referring clinic, 56 cats (57.7%) were
reported to have a single mass, 40 (40.8%) had multiple
masses, and the number of masses was unreported in 1. A total
of 43 cases (43.9%) were classified as WHO stage 1, 15
(15.3%) as stage 2, and 32 (33%) as stage 3, with 7 (7.2%)
unknown (survey information incomplete).
Results of Kaplan-Meier survival analysis of survey data
and histologic features are listed in Tables 3, 4, and 5. Although
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 diameter. More advanced WHO stage and the presence of lymph
node metastases also showed statistically significant negative
correlation with OS (both P ¼ .01). Both WHO histologic subtype (P ¼ .005) and the presence of lymphovascular invasion
(P < .001) corresponded with OS. A weak association was
found between tumors with >75% tubule formation and those
with less than 10% tubule formation (P ¼ .037) and OS, the
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
Mills et al
243
Table 2. Novel Grading System for Evaluation of Invasive Mammary Carcinoma in Female Cats.
Histologic Featurea
Score
Lymphovascular invasion
Absent
Present
Nuclear formb
5% abnormal
>5% abnormal
Mitotic countc
62
>62
0
1
0
1
0
1
Total Score
Grade
0
1
2–3
I (low-grade carcinoma)
II (intermediate-grade carcinoma)
III (high-grade carcinoma)
Median Survival, mo
Survival at 18 mo, %
31
14
8
82
37
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)
Age (median, 11 y)
(i) 11 y
(ii) > 11 y
Reproductive status
(i) Intact
(ii) Spayed
Tumor diameter
(i) <2 cm
(ii) 2–3 cm
(iii) >3 cm
Lymph node metastases
(i) No
(ii) Yes
WHO stage
(i) 1
(ii) 2
(iii) 3
50 (15)
47 (14)
15 (11)
82 (15)
51 (16)
18 (14)
19 (11)
66 (16)
17 (9)
43 (18)
15 (15)
32 (10)
P Value
Hazard Ratio
95% CI
.9
—
—
.2
—
—
.09
.17b
.49c
.046d
.01
—
—
.01
.19b
.20c
.004d
1
—
—
1.5
—
—
0.66–1.6
—
—
0.8–2.9
—
—
0.65b
0.78c
0.50d
0.39
—
—
0.35–1.2
0.38–1.6
0.25–1.0
0.19–0.8
—
—
0.63b
0.66c
0.43d
0.31–1.3
0.35–1.3
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
cats with solid or cribriform carcinomas (10 and 8 months,
respectively) was less than half that of cats with the tubulopapillary subtype (21 months). Percentage of necrosis, the presence or nature of any inflammation, the stromal response,
and the percentage of squamous differentiation were not statistically significant with respect to OS. In contrast, with the
exception of chromatin vesiculation, all nuclear characteristics
evaluated within the neoplastic population were significantly
and inversely related to OS, including abnormal nucleolar morphology (P ¼ .013), increased anisokaryosis (P ¼ .013),
increased size (P ¼ .038), abnormal nuclear form (P < .001),
increased nuclear pleomorphism score (P ¼ .006), and
increased mitotic count (P ¼ .021). Visible nucleoli were
absent in only 2 tumors (1.9%); nuclear size was increased relative to normal in 104 tumors (96.3%). The mean mitotic count
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
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)
Tumor subtype
(i) Tubulopapillary
(ii) Solid
(iii) Cribriform
(iv) Squamous cell carcinoma
Tubule formatione
(i) <10%
(ii) 10%–75%
(iii) >75%
Necrosis
(i) 25%
(ii) >25%
Inflammation
(i) None present or mild
(ii) Lymphoplasmacytic
(iii) Neutrophilic or pleocellular
Lymphovascular invasion
(i) Absent
(ii) Present
Stromal response
(i) Normal (mild-moderate)
(ii) Marked
(iii) Intratumoral
Squamous differentiation
(i) 5%
(ii) >5%
61 (21)
36 (10)
10 (8)
1 (—)
37 (13)
54 (14)
17 (29)
56 (18)
52 (12)
34 (18)
59 (15)
15 (9)
68 (18)
40 (8)
71 (14)
29 (14)
8 (31)
67 (15)
41 (12)
P Value
.005
<.001b
.95c
.043d
—
.1
.45b
.08c
.037d
.1
—
—
.22
.23b
.33c
.15d
<.001
—
—
.16
.15b
.61c
.13d
.2
—
—
Hazard Ratio
95% CI
0.41b
.98c
.32d
—
0.24–0.68
0.42–2.2
0.10–0.97
—
1.2b
1.7c
2.0d
0.69
—
—
0.74–1.9
0.93–3.2
1.0–3.7
0.44–1.1
—
—
0.74b
0.71c
0.57d
0.38
—
—
0.45–1.2
0.36–1.4
0.27–1.2
0.23–0.62
—
—
1.4b
1.3c
1.8d
0.74
—
—
0.88–2.3
0.48–3.5
0.84–3.8
0.46–1.2
—
—
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
value of 62. When divided into tertiles, categories were 0 to 50,
51 to 70, and >71 mitotic figures per 10 high-power fields. In
contrast, when mitotic counts were grouped according to the
EE grading system, 1 tumor (1.0%) was given a score of 1, 4
(3.7%) a score of 2, and 103 (95.4%) a score of 3.
Only lymphovascular invasion, mitotic count, and nuclear
form could be considered independent prognostic factors in the
current study (P ¼ .008, .004, and <.001, respectively) according to Cox proportional hazards analysis (Suppl. Table S1).
Overall, EE grading was not associated with OS (Fig. 5). A statistically significant difference was found between grades II and
III (P ¼ .026), with a median survival of 17 months (n ¼ 63) and
13 months (n ¼ 43), respectively. Only 2 tumors (1.9%) received
a nuclear pleomorphism score of 1, and only 5 (4.6%) received a
mitotic count score of either 1 or 2 (data not shown).
A strong association was found between the MMEE grading
and OS (P < .001; Fig. 6), REE grading and OS (P < .001; Fig.
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
I carcinomas (1.9%) using the original EE grading system
(median survival 19 months). Median survival was 14 months
(n ¼ 61) and 13 months (n ¼ 18) for cats with grade II and III
carcinomas, respectively. REE grading resulted in 32 carcinomas (29.6%) being classified as grade I (median survival 29
months). This was 2.5-fold longer OS than grade II carcinomas
(median survival 12 months; n ¼ 63) and 5.8-fold longer
OS than grade III carcinomas (median survival 5 months;
n ¼ 13). When individual grades were analyzed separately, a
statistically significant difference was not identified between
grades II and III in the MMEE or the REE grading system.
When tumors were graded histologically using the novel system, a statistically significant difference was present between
grades I and II (P ¼ .0012), grades II and III (P ¼ .024), and
grades I and III (P < .0001). Cats with grade III (high-grade)
carcinomas had only one-fourth (25.8%) the survival time of
cats with grade I (low-grade) carcinomas (median survival
8 months [n ¼ 48] and 31 months [n ¼ 22], respectively) and
less than two-thirds (57%) the median survival time of cats
with grade II carcinomas (median survival 14 months; n ¼ 38).
Discussion
To our knowledge, this is the largest histologic analysis of
feline mammary carcinoma to date, aimed at evaluating and
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
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)
Chromatin vesiculation
(i) Absent or mild
(ii) Moderate
(iii) Marked
Nucleoli
(i) Indistinct
(ii) Single and small
(iii) Abnormal and/or multiple
Anisokaryosis
(i) <2-fold variation
(ii) 2- to 3-fold variation
(iii) >3-fold variation
Nuclear size
(i) Normal
(ii) 2-fold larger than normal
(iii) >2-fold larger than normal
Nuclear form
(i) 5% abnormal
(ii) 5%–25% abnormal
(iii) >25% abnormal
Nuclear pleomorphism scoree
(i) 1
(ii) 2
(iii) 3
Mitotic countf (median, 62)
(i) 62
(ii) >62
71 (13)
34 (15)
3 (18)
2 (34)
96 (15)
10 (4)
69 (18)
35 (9)
4 (14)
4 (35)
89 (14)
15 (16)
64 (21)
36 (12)
8 (12)
2 (34)
83 (16)
23 (11)
54 (18)
54 (9)
P Value
.96
.99b
.71c
.84d
.013
.58b
.004c
.43d
.013
.048b
.8c
.18d
.038
.027b
.35c
.002d
<.001
<.001b
.85c
.12d
.006
.68b
.002c
.21d
.021
—
—
Hazard Ratio
95% CI
1.0b
0.78c
0.88d
0.62–1.6
0.20–3.0
0.26–3.0
0.65b
0.2c
0.52d
0.14–3.0
0.07–0.59
0.1–2.7
0.47b
1.1c
0.38d
0.28–0.8
0.42–3.1
0.1–1.5
0.38b
0.73c
0.14d
0.16–0.89
0.38–1.4
0.04–0.48
0.35b
1.1c
0.45d
0.2–0.61
0.48–2.4
0.16–1.2
0.71b
0.38c
0.43d
0.59
—
—
0.14–3.7
0.2–0.69
0.12–1.6
0.37–0.92
—
—
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 5. Kaplan-Meier curve depicting Elston and Ellis grade (Table
1) vs overall survival in female cats with invasive mammary carcinoma.
Median survival was 19 months, 17 months, and 13 months for grade I,
II, and III tumors, respectively. Hashmarks indicate censored events.
Figure 6. Kaplan-Meier curve depicting mitotic-modified Elston and
Ellis grade (Table 1) vs overall survival in female cats with invasive
mammary carcinoma. Median survival was 27 months, 14 months, and
13 months for grade I, II, and III tumors, respectively. Hashmarks indicate censored events.
improving current grading methodologies. Due to the current
lack of a globally accepted, reliable, species-specific grading
system, the poor prognosis generally conferred upon cats with
malignant mammary carcinomas in diagnostic practice is not
typically revised as a result of histologic assessment. It was evident in this and previous studies, however, that survival times
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
246
Veterinary Pathology 52(2)
Figure 7. Kaplan-Meier curve depicting revised Elston and Ellis grade
(Table 1) vs overall survival in female cats with invasive mammary carcinoma. Median survival was 29 months, 12 months, and 5 months for
grade I, II, and III tumors, respectively. Hashmarks indicate censored
events.
Figure 8. Kaplan-Meier curve depicting novel grade (Table 2) vs overall survival in female cats with invasive mammary carcinoma. Median
survival was 31 months, 14 months, and 8 months for grade I, II, and
III tumors, respectively. Hashmarks indicate censored events.
in affected cats are variable, with postsurgical survival extending for years rather than months in many cases.19,29,36 Findings
of this study support the use of modified EE grading systems or
a novel grading system to improve the prognostic value of histologic grading for feline mammary carcinoma.
Tumor size (or diameter) is easily estimated at presentation
to a primary practice and is a commonly used diagnostic indicator due to its reported correlation with prognosis in feline
mammary carcinoma.14,16,39 Our study demonstrated only a
weak statistical association between tumor diameter and OS
and only for tumors greater than 3 cm in diameter. This may
be due to insufficient numbers as well as the inherent subjectivity involved in measurement of tumor diameter (calipers vs palpation, in vivo vs ex vivo, etc). Additional shortcomings and
possible sources of error encountered when using tumor size
(or volume) as a prognostic factor are described by Matos
et al.18 Recent reports support findings here, indicating that
while larger tumors (>3 cm diameter) are associated with
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 prognostic 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 staging 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 population resulted in inferior discrimination between tumors and
lacked overall association with OS. Tumors were heavily
skewed toward grade II or III (58.3% and 39.8% of carcinomas, respectively), resulting in underrepresentation of grade I
(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 investigation by Seixas et al34 is most comparable to the present
study in both scope and objective. Similar to our findings, only
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
explanations for the discrepant results found in this study
include the inherent subjectivity associated with tumor grading,23 differences in population-specific factors (ie, spayed vs
intact, geographic location), differences attributable to study
type and case definition, or an inherently deficient grading
system.
To address the latter possibility, numerous morphological
criteria were evaluated in the present study and analyzed with
respect to OS. Tumor subtypes were included, despite their
characterization as descriptive rather than prognostic.20 Indeed,
a significant association between tumor subtype and OS was
found here and by Seixas et al34 but not by Millanta et al19
or Castagnaro et al.4 This discrepancy may reflect the inherent
subjectivity of histologic evaluation, the morphological heterogeneity of feline mammary carcinoma, or variable interpretation of the category descriptions.20
Both the mitotic count and the presence of lymphovascular
invasion demonstrated a strong correlation with OS, similar to
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
Mills et al
247
findings in previous studies.29,34 Mitotic index and histologic
staging based on lymphovascular invasion were both found
to be independent prognostic factors by Preziosi et al.29 The
prognostic significance of invasion was also recognized by
Mandelli et al,17 who developed a histologic grading system for
feline mammary gland tumors based on this feature alone.
In this study, nucleolar morphology, anisokaryosis, nuclear
size, nuclear form, and nuclear pleomorphism score all showed
a significant correlation with OS in univariate analysis. Upon
multivariable analysis, however, nuclear form was the only
aspect of nuclear morphology shown here to be an independent
prognostic factor in feline mammary carcinoma. Nuclear form
was previously evaluated using nuclear morphometry software.
DeVico and Maiolino6 concluded that both the standard deviation and coefficient of variation of nuclear form were reliable
prognostic factors in cats with mammary carcinoma. In a more
recent study, however, Simeonov and Simeonova35 found no
statistical correlation between nuclear morphometric parameters and OS upon analysis of cytological samples from feline
mammary carcinomas. Further investigation is warranted given
the results of the present study.
In light of the multivariate analysis results together with the
apparent shortcomings of EE grading when applied to the study
population, 3 new grading systems were developed in an
attempt to improve histologic grading of feline mammary carcinoma. First, as has been recently described for grading canine
mammary carcinomas,27 modifications of the EE grading system were proposed. The MMEE grading system features
increased range subcategories within the mitotic count
category to better accommodate the high median and broad
range of mitotic counts encountered within our study population. Application of the MMEE grading system greatly
improved detection of grade I (well-differentiated) carcinomas
compared with the EE grading system, both in terms of the
number of tumors recognized and median survival. The REE
grading system represents a more extensive modification of
the EE grading system to further improve tumor discrimination. Nuclear pleomorphism, a significant source of skewness
within the data and long recognized as the most subjective criterion in the EE system,7 was removed and replaced by
nuclear form. Although still admittedly subjective, this allows
the diagnostic pathologist to concentrate on a single aspect of
nuclear morphology irrespective of concurrent nuclear features. Lymphovascular invasion was also added, recognizing
its prognostic significance. REE grading again resulted in
superior recognition of grade I (well-differentiated) carcinomas compared with EE grading. In addition, grade III (poorly
differentiated) carcinomas were more adequately represented
in terms of median survival. It should be noted, however, that
neither the MMEE nor the REE grading system was able to
show significant differences between grades II and III. In the
latter case, this may be attributable to the low number of grade
III carcinomas.
In practical terms, grading tumors with the MMEE grading
system is virtually identical to using the original EE grading
system. Potential drawbacks of the REE grading system
include the additional time required for evaluation of a fourth
criterion and familiarization with nuclear form scoring.
A novel grading system was also developed, based solely on
those criteria found to be independent prognostic factors: lymphovascular invasion, mitotic count, and nuclear form. Only
2 subcategories were established for mitotic count and nuclear
form to simplify the grading system. When applied to the study
population, a statistically significant difference was found
between all grades in terms of OS. Although fewer were identified relative to MMEE and REE grading, grade I (low grade)
carcinomas were well represented. The presence of only 2 categories associated with each criterion greatly simplifies histologic scoring using this system, increasing the potential utility for
the diagnostic pathologist. In addition, cats with grade I (lowgrade) carcinomas were again more adequately represented by
the novel grading system compared with the EE grading
system.
A potential limitation associated with use of lymphovascular invasion in the REE and novel grading systems is the inconsistent presence between histologic sections of the same tumor.
While it is true that the extent to which identifying lymphovascular invasion depends on which portion(s) or serial section(s)
of the tumor are evaluated, the impact this has on its value as a
prognostic indicator is unknown. There is suboptimal sensitivity associated with many histologic criteria evaluated on a 2dimensional section of a 3-dimensional, heterogeneous tumor.
Despite these potential shortcomings, vascular invasion has
been included in histologic grading systems for other tumors,
including squamous cell carcinoma of the tongue in dogs,3 as
well as for prognostication in feline primary lung carcinomas.10
In contrast to dogs, where the protective effect of early
ovariohysterectomy on the development of mammary carcinoma is well established, less is known about the impact
of reproductive hormones on feline mammary carcinoma.
Overley et al26 demonstrated a clear protective effect of ovariohysterectomy, whereby those cats having undergone the
procedure prior to 1 year of age showed an 86% reduction
in risk of mammary carcinoma relative to intact cats. Unfortunately, scant information is available regarding possible
effect(s) of spay status on tumor characteristics, grading, and
OS in cats with mammary carcinoma. No difference was
found between spayed and intact cats with regard to OS in the
present study, although median survival was 66% higher in
the former group. This may simply be due to a lack of statistical power, as only 15% (n ¼ 16) of carcinomas were from
intact animals. In dogs with mammary carcinoma, spay status
has been shown to be significantly associated with survival.5,27 Recently, Hughes and Dobson13 called attention to
the need for larger studies to investigate the effect of spay status on prognostic marker expression in feline mammary carcinomas. It is currently unknown to what extent the effects of
endogenous estrogen and/or progesterone exposure may manifest histologically and/or immunohistologically in feline
mammary carcinoma. If present, this may influence diagnostic evaluation, including any attempts at histologic grading.
Further investigation is required in this area.
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
248
Veterinary Pathology 52(2)
One of the strengths of the present study is that most tumor
biopsy specimens originated from primary practices in rural,
semi-rural, and urban areas rather than large referral centers,
reducing bias and improving the generalizability of results. The
primary limitation of the present study is its retrospective and
multi-institutional nature. Patient data acquired from surveys may
have been incomplete or inaccurate, and there was a clear bias
favoring available specimens. Furthermore, recordkeeping, diagnostic workup, sample collection/processing, and diagnosis lack
standardization in such studies.37 Except in rare cases, no postmortem examinations were carried out to confirm the extent of
disease or cause of death. In some cases, multiple masses were
reported by the primary practitioner, but not all were submitted.
Every attempt was made to include all potential primary tumors
as recommended by Matos et al,18 although it remains unknown
whether unsubmitted tumors were in fact primary, higher grade,
or mammary in origin and to what extent they influenced OS.
In summary, the use of the EE grading system for feline mammary carcinoma has not been adopted widely by diagnostic
pathologists because it was developed for human breast cancer,
it is time-consuming, and its use is not perceived to improve upon
the generally poor prognosis associated with feline carcinomas.
Application of the EE grading system in our study population
resulted in inferior discrimination between tumors with respect
to OS. Species-specific, reliable histologic grading of feline mammary carcinomas has the potential to significantly improve routine diagnostic evaluation and prognostication. Currently, no
such grading system exists, although the MMEE, REE, and the
novel grading systems introduced here may represent a step in this
direction. All 3 significantly improved the prognostic value of histologic grading for feline mammary carcinoma, especially for
the subset of affected cats exhibiting prolonged survival that may
otherwise go unrecognized. Finally, while the results and proposed grading systems presented herein are applicable to the
study population, it is unclear to what extent they are applicable
to the greater feline population. Future studies are required to validate these findings, elucidate the role of spay status if any, and
provide further guidance as to the most appropriate grading system to use in female cats with mammary carcinoma.
Acknowledgements
We thank Phil Dillman and the Histology Laboratory as well as
Melissa Koehnlein and Dale Godson in the Immunology Laboratory
at Prairie Diagnostic Services, Inc for their expertise.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship and/or publication of this article: This work
was supported by the WCVM Companion Animal Health Fund.
References
1. Baak JPA, Gudlaugsson E, Skaland I, et al. Proliferation is the strongest prognosticator in node-negative breast cancer: significance,
error sources, alternatives and comparison with molecular prognostic markers. Breast Cancer Res Treat. 2009;115(2):241–254.
2. Burrai GP, Mohammed SI, Miller MA, et al. Spontaneous feline
mammary intraepithelial lesions as a model for human estrogen
receptor– and progesterone receptor–negative breast lesions.
BMC Cancer. 2010;10:156.
3. Carpenter LG, Withrow SJ, Powers BE, et al. Squamous cell carcinoma of the tongue in 10 dogs. J Am Anim Hosp Assoc. 1993;
29(1):17–24.
4. Castagnaro M, Casalone C, Bozzetta E, et al. Tumour grading and
the one-year post-surgical prognosis in feline mammary carcinomas. J Comp Pathol. 1998;119(3):263–275.
5. Chang SC, Chang CC, Chang TJ, et al. Prognostic factors associated with survival two years after surgery in dogs with malignant
mammary tumors: 79 cases (1998–2002). J Am Vet Med Assoc.
2005;227(10):1625–1629.
6. DeVico G, Maiolino P. Prognostic value of nuclear morphometry in
feline mammary carcinomas. J Comp Pathol. 1997;117(2):99–105.
7. Dunne B, Going JJ. Scoring nuclear pleomorphism in breast cancer. Histopathology. 2001;39(3):259–265.
8. Elston CW, Ellis IO. Pathologic prognostic factors in breast cancer, I: the value of histologic grade in breast cancer: experience
from a large study with long-term follow-up. Histopathology.
1991;19(5):403–410.
9. Gimenez F, Hecht S, Craig LE, et al. Early detection, aggressive
therapy optimizing the management of feline mammary masses. J
Feline Med Surg. 2010;12(3):214–224.
10. Hahn KA, McEntee MF. Prognosis factors for survival in cats
after removal of a primary lung tumor: 21 cases (1979–1994). Vet
Surg. 1998;27(4):307–311.
11. Hayes AA, Mooney S. Feline mammary tumors. Vet Clin North
Am Small Anim Pract. 1985;15(3):513–520.
12. Hayes HM Jr, Milne KL, Mandell CP. Epidemiological features of
feline mammary carcinoma. Vet Rec. 1981;108(22):476–479.
13. Hughes K, Dobson JM. Prognostic histopathological and molecular
markers in feline mammary neoplasia. Vet J. 2012;194(1):19–26.
14. Ito T, Kadosawa T, Mochizuki M, et al. Prognosis of malignant mammary tumor in 53 cats. J Vet Med Sci. 1996;58(8):
723–726.
15. Lana S, Rutteman GR, Withrow SJ. Tumors of the mammary
gland. In: Withrow SJ, Vail DM, eds. Withrow and Macewan’s
Small Animal Clinical Oncology. 4th ed. Philadelphia, PA: Saunders Elsevier; 2007:619–636.
16. Macewen EG, Hayes AA, Harvey HJ, et al. Prognostic factors for
feline mammary tumors. J Am Vet Med Assoc. 1984;185(2):
201–204.
17. Mandelli G, Scanziani E, Cairoli F. ‘‘Grading’’ istologico delle
neoplasie mammarie dei carnivori. La Clinica Veterinaria.
1987;110:347–357.
18. Matos AJF, Baptista CS, Gartner MF, et al. Prognostic studies of
canine and feline mammary tumours: the need for standardized
procedures. Vet J. 2012;193(1):24–31.
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016
Mills et al
249
19. Millanta F, Lazzeri G, Mazzei M, et al. Mib-1 labeling index in
feline dysplastic and neoplastic mammary lesions and its relationship with postsurgical prognosis. Vet Pathol. 2002;39(1):120–126.
20. Misdorp W, Else RW, Hellmen E, et al. Histological Classification of Mammary Tumors of the Dog and the Cat. Second series,
vol vii. Armed Forces Institute of Pathology; 1999.
21. Misdorp W, Romijn A, Hart AA. Feline mammary tumors: a casecontrol study of hormonal factors. Anticancer Res. 1991;11(5):
1793–1797.
22. Misdorp W, Weijer K. Animal model: feline mammary carcinoma. Am J Pathol. 1980;98(2):573–576.
23. Northrup NC, Harmon BG, Gleger TL, et al. Variation among
pathologists in histologic grading of canine cutaneous mast cell
tumors. J Vet Diagn Invest. 2005;17(3):245–248.
24. Novosad CA. Principles of treatment for mammary gland tumors.
Clin Tech Small Anim Pract. 2003;18(2):107–109.
25. Novosad CA, Bergman PJ, O’Brien MG, et al. Retrospective evaluation of adjunctive doxorubicin for the treatment of feline mammary gland adenocarcinoma: 67 cases. J Am Anim Hosp Assoc.
2006;42(2):110–120.
26. Overley B, Shofer FS, Goldschmidt MH, et al. Association
between ovarihysterectomy and feline mammary carcinoma. J
Vet Intern Med. 2005;19(4):560–563.
27. Pena L, De Andres PJ, Clemente M, et al. Prognostic value of histological grading in noninflammatory canine mammary carcinomas in a prospective study with two-year follow-up:
relationship with clinical and histological characteristics. Vet
Pathol. 2013;50(1):94–105.
28. Petterino C, Ratto A, Arcicasa E, et al. Expression of stat3 in
feline mammary gland tumours and its relation to histological
grade. Vet Res Commun. 2006;30(6):599–611.
29. Preziosi R, Sarli G, Benazzi C, et al. Multiparametric survival
analysis of histological stage and proliferative activity in feline
mammary carcinomas. Res Vet Sci. 2002;73(1):53–60.
30. Rakha EA, El-Sayed ME, Lee AHS, et al. Prognostic significance
of Nottingham histologic grade in invasive breast carcinoma. J
Clin Oncol. 2008;26(19):3153–3158.
31. Rakha EA, Reis JS, Baehner F, et al. Breast cancer prognostic
classification in the molecular era: the role of histological grade.
Breast Cancer Res. 2010;12:207.
32. Rasotto R, Caliari D, Castagnaro M, et al. An immunohistochemical study of her-2 expression in feline mammary tumours.
J Comp Pathol. 2011;144(2–3):170–179.
33. Ressel L, Millanta F, Caleri E, et al. Reduced PTEN protein
expression and its prognostic implications in canine and feline
mammary tumors. Vet Pathol. 2009;46(5):860–868.
34. Seixas F, Palmeira C, Pires MA, et al. Grade is an independent
prognostic factor for feline mammary carcinomas: a clinicopathological and survival analysis. Vet J. 2011;187(1):65–71.
35. Simeonov R, Simeonova G. Nuclear cytomorphometry in feline
mammary gland epithelial tumours. Vet J. 2009;179(2):296–300.
36. Viste JR, Myers SL, Singh B, et al. Feline mammary adenocarcinoma: tumor size as a prognostic indicator. Canadian Vet J. 2002;
43(1):33–37.
37. Webster JD, Dennis MM, Dervisis N, et al. Recommended guidelines for the conduct and evaluation of prognostic studies in veterinary oncology. Vet Pathol. 2011;48(1):7–18.
38. Weijer K, Hart AAM. Prognostic factors in feline mammary carcinoma. J Natl Cancer Imst. 1983;70(4):709–716.
39. 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.
1972;49(6):1697–1704.
40. Yoshimura H, Michishita M, Ohkusu-Tsukada K, et al. Appearance
and distribution of stromal myofibroblasts and tenascin-C in feline
mammary tumors. Histol Histopathol. 2011;26(3):297–305.
41. Zappulli V, De Zan G, Cardazzo B, et al. Feline mammary tumours
in comparative oncology. J Dairy Res. 2005;72:98–106.
Downloaded from vet.sagepub.com at PENNSYLVANIA STATE UNIV on May 16, 2016