Original Article

Veterinary Pathology
1-11
Feline Invasive Mammary Carcinomas: ª The Author(s) 2019
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Prognostic Value of Histological Grading DOI: 10.1177/0300985819846870
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Elie Dagher1, Jérôme Abadie1,2, Delphine Loussouarn2,3,

Mario Campone2,4, and Frédérique Nguyen1,2,4

Abstract
Feline mammary carcinomas are highly malignant tumors usually associated with poor outcome. Nevertheless, survival times can
differ significantly according to various prognostic factors. The Elston and Ellis (EE) histologic grading system, originally developed
for human breast cancer, is commonly used to grade feline mammary carcinomas, although it is not really adapted for this species,
hence the need of a more relevant grading system. Although few veterinary studies attempted to validate previously published
results in an independent cohort, the aim of our study was to evaluate the prognostic value of different histologic grading systems
in feline invasive mammary carcinomas, including the EE grading system applicable to human breast cancers and the modified and
newly designed histologic grading systems recently proposed by Mills et al. Survey data and histologic features of 342 feline
invasive mammary carcinomas were analyzed with respect to overall and cancer-specific survival. The histological grading system
with best prognostic value was the mitotic-modified Elston and Ellis (MMEE) grading system: grade III carcinomas (P ¼ .04, hazard
ratio [HR] ¼ 1.46, 95% CI, 1.01–2.11), grade II (P ¼ .03, HR ¼ 1.39, 95% CI, 1.03–1.88), and grade I carcinomas (HR ¼ 1.00,
reference), with decreasing hazard ratios significantly were associated with a worse overall survival, independently from the
pathologic tumor size (pT  20 mm: P ¼ .002, HR ¼ 1.45, 95% CI, 1.15–1.83) and positive nodal stage (P ¼ .001, HR ¼ 1.51, 95%
CI, 1.18–1.94). This retrospective study validates Mills et al’s proposal to adapt the thresholds for mitotic counts to better assess
the histological grade of the highly proliferative mammary carcinomas encountered in the cat.

Keywords
cats, Elston and Ellis histologic grade, histological grading system, mammary carcinoma, oncology, prognostic factor, survival

Mammary tumors are common in cats and constitute approxi- and is based on 3 parameters: tubule formation, nuclear pleo-
mately 17% of all feline neoplasms,7,16 with reported malig- morphism, and mitotic count.5 According to Castagnaro et al,2
nancy rates ranging between 80% and 90%.6,9 Feline mammary the Elston and Ellis (EE) grading system has good prognostic
carcinomas (FMCs) have a tendency to be biologically aggres- value for well-differentiated (grade I) and poorly differentiated
sive, with a median reported overall survival time of 8 to 12 (grade III) feline mammary carcinomas but not grade II tumors.
months postdiagnosis in most studies with follow-up.2–4,11,23 The same grading method was found to have high prognostic
However, survival times can differ significantly according to value in queens with invasive carcinomas by Millanta et al.14
various prognostic factors, reviewed by Zappulli et al31 and Seixas et al23 found the EE grading system to be a significant
others,6 such as the pathologic tumor size,8,10,23,26,29 lympho- prognostic factor by multivariate analysis for middle-aged to
vascular invasion,19,21,23,28,29 the presence of nodal or distant elderly queens with mammary carcinomas, independent from
metastasis,23,29 the clinical stage according to the World Health lymphovascular invasion in terms of disease-free survival and
Organization (WHO),8 the histological type,22–25 histological
grade,2,12,14,15,23 margin status,29 skin ulceration,29 peritumoral
1
lymphoplasmacytic inflammation,29 and some immunohisto- AMaROC, Oniris (Nantes Atlantic College of Veterinary Medicine, Food
Science and Engineering), France
chemical markers such as cyclooxygenase-2 expression,13 the 2
CRCINA, INSERM, Université d’Angers, Université de Nantes, Nantes,
Ki-67 proliferation index,4,20,23,26 and the molecular subtypes France
defined by estrogen receptor alpha (ER), progesterone receptor 3
Department of Pathology, University hospital, Nantes, France
4
(PR), the feline homolog of human epidermal growth factor ICO, Integrated Center for Oncology Nantes, Angers, France
receptor-2 (HER2), Ki-67, and cytokeratins 5 and 6 (CK5/6)
Corresponding Author:
expression.26,27 Frédérique Nguyen, AMaROC, Oniris site Chantrerie, CS40706, cedex 3,
The most widely accepted grading system for FMCs was Nantes 44307, France.
adapted from the grading system used in human breast cancer Email: [email protected]
2 Veterinary Pathology XX(X)

independent from lymph node metastasis in terms of overall evident at time of diagnosis, the animal was treated solely by
survival. These studies present persuasive evidence in support surgery (no chemotherapy or radiation therapy pre- or postmas-
of histologic grading using the EE grading system as a predic- tectomy), there was no evidence of distant metastasis at diag-
tor of overall survival in cats with FMCs. nosis, and follow-up was available for at least 48 months
Recently, Mills et al15 reevaluated the classic EE grading postsurgery.
system that failed to correlate significantly with cancer-specific Exclusion criteria included patients with noninvasive in situ
survival in their study. Using multivariate analysis, they found mammary carcinomas, those who had presented another malig-
that lymphovascular invasion, nuclear form, and mitotic count nant tumor than the mammary carcinoma either before or at the
each demonstrated independent prognostic significance regard- time of diagnosis, the animals whose outcome was unknown
ing cancer-specific survival of cats with invasive mammary after the diagnosis of mammary carcinoma, cats treated with
carcinomas. Modifications of the EE system and a novel grad- adjuvant chemotherapy or radiation therapy, stage IV disease,
ing system were then proposed, and all showed significant and the carcinomas that were improper for adequate immuno-
correlation with cancer-specific survival.15 histochemical staining. A total of 342 female cats with stages
Few veterinary studies attempted to validate previously pub- I–III invasive mammary carcinoma were included in this study.
lished results in an independent cohort. Therefore, the aim of
our study was to evaluate the prognostic value of different
Processing of Tissues and Immunohistochemistry for
histologic grading systems, including the EE grading system
applicable to human breast cancers, and the modified and Histologic Staging Purposes
newly designed histologic grading systems recently proposed The tumor samples were fixed in 10% formalin and embedded
by Mills et al.15 for feline invasive mammary carcinomas in an in paraffin, cut into 3 mm–thick sections, and then stained with
independent retrospective cohort of 342 female cats with stages hematoxylin-eosin-saffron (HES). Automated immunohisto-
I–III invasive mammary carcinoma. chemistry (Benchmark XT Ventana Medical Systems, Roche
Diagnostics) was used on 3 mm–thick sections for confirmation
of lymphovascular invasion using LMO2 as a lymphatic
Materials and Methods endothelium marker (LIM domain-only protein-2, clone
SP51, Spring M351) and evaluation of invasiveness using
Case Origin and Data Collection p63 as a myoepithelial cell marker (clone 4A4, Abcam
This retrospective study included 342 cases of feline mammary ab735). Mammary carcinomas limited by a continuous layer
carcinomas that were diagnosed between 2007 and 2010 in 2 of p63-positive myoepithelial cells were defined as mammary
laboratories for veterinary diagnostic pathology: the Labora- carcinomas in situ and excluded from the present study. Mam-
toire d’Histopathologie Animale (LHA) at Oniris, Nantes, and mary carcinomas with a focal interruption of their p63-positive
the Laboratoire d’Anatomie Pathologique Veterinaire (LAPV) myoepithelial cell lining, less than 1 mm long, were considered
of Amboise, France. The owners’ written consent and approval micro-invasive and also excluded from the present study.
from the local animal welfare committee of Oniris were Mammary carcinomas lacking a p63-positive myoepithelial
obtained prior to inclusion. Collected information regarding cell layer over at least 1 mm of their circumference were
the cases included signalment (age, breed, spaying status), his- defined as invasive and were included. This technique was used
tory of contraception and parity, and medical history. only when invasiveness was doubtful on HES-stained slides.
Tumors were staged according to the modified WHO sta-
ging system.17,18 Stage I included cats with primary tumors less Histologic Assessment Criteria Except Histological Grade
than 20 mm of largest diameter on histological sections (patho-
logic tumor size), with no evidence of regional or distant In case of multiple (within a given mammary gland) or multi-
metastases; stage II included cats with tumors 20 to 30 mm centric (within different mammary glands) invasive mammary
in diameter on histological sections, with no evidence of carcinomas, the carcinoma with the largest diameter on histo-
regional or distant metastases. Stage III included cats with logical section was selected for analysis. The histological types
primary tumors less than 30 mm of tumor size, with evidence included tubulopapillary (tubular, papillary, and tubulopapil-
of regional metastases, or cats with tumors greater than 30 mm lary), solid (including adenosquamous, squamous cell, and ana-
in diameter, with or without evidence of regional metastases plastic), cribriform (containing small hole-like lumens within a
but without evidence of distant metastases. Stage IV FMCs solid background), and mucinous, with most of the cases
(with evidence of distant metastases at diagnosis, regardless demonstrating more than 1 growth pattern. In these instances,
of tumor size or regional metastases) were excluded from the the less differentiated pattern of the tumor (ie, anaplastic or
present study. solid) determined the subtype; or when 2 patterns that could
be considered equally well differentiated coexisted (ie, cribri-
form and mucinous), the largest growth pattern determined the
Inclusion/Exclusion Criteria subtype.
Feline patients were eligible for inclusion when an invasive The pathologic tumor size (pT) was measured on HES-
mammary carcinoma was the only primary malignant tumor stained histological sections as the largest tumor dimension
Dagher et al 3

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

Applicable Grading System Histologic Feature Score

EE, MMEE, REE Tubule formation (% of tumor area)

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 (least differentiated/most invasive portions)
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
0–10 0–33 1
11–19 34–66 2
20 67 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 (according to Mills et al15)
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 0.625 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. 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.

in millimeters. Lymphovascular invasion (LVI), the presence inflammation had to contain nodular lymphohistiocytic infil-
of lymph and/or blood vessel emboli, was assessed on HES- trates resembling lymphoid follicles.
stained slides and confirmed by LMO2 immunohistochemistry
in case of doubt. Positive lymphovascular invasion was con-
firmed if neoplastic emboli were clearly seen within an LMO2-
EE: Elston and Ellis Histologic Grading System
positive row of lymphatic endothelial cells. Central necrosis of
any type (coagulative, lytic, or comedonecrosis) affecting At first, tumors were graded according to the EE grading sys-
groups of neoplastic cells (but not single scattered cells) was tem described for human breast cancer (Table 1),5 where car-
assessed as present/absent regardless of extension. Squamous cinomas were scored according to 3 criteria: the percentage of
differentiation affecting groups of viable neoplastic cells (not tubule formation, subjectively assessed at low-power magnifi-
directly adjacent to necrotic foci), manifested by an eosinophi- cation and quantified as a percentage of the tumor parenchyma;
lic glassy appearance of the cytoplasm, not necessarily with the degree of nuclear pleomorphism, assessed at high power
keratin pearl formation, was assessed as present/absent regard- (400) magnification in the least differentiated and/or most
less of extension. Peritumoral tumor-associated lymphoplas- invasive portion of the tumor, typically along the periphery;
macytic and macrophagic inflammation was quantified as and the mitotic count in 10 high-power fields (400). A total
absent (0), minimal (1), mild (2), moderate (3), marked (4), score was calculated for each tumor by summing the points of
or severe (5) and then considered negative for scores 0 to 2 the 3 categories to determine the grade (I: 3–5 points, II: 6 or 7
and positive for scores 3 to 5. To be considered moderate, points, or III: 8 or 9 points), indicating respectively well-
peritumoral mononuclear inflammation had to involve at least differentiated, moderately differentiated, or poorly differen-
half of the circumference of the mammary carcinoma, marked tiated carcinomas.
peritumoral inflammation had to be multifocally observed Three grading systems were then applied to our cohort using
along all the circumference, and severe peritumoral the grading systems designed by Mills et al.15
4 Veterinary Pathology XX(X)

MMEE: Mitotic-Modified Elston and Ellis Histologic

Grading System
In the mitotic-modified Elston and Ellis (MMEE) grading sys-
tem proposed by Mills et al15 (Table 1), mitotic figures were
counted in 10 consecutive fields at the periphery of the tumor in
the areas of highest proliferative activity. The mitotic count
categories of the EE grading system were modified to better
accommodate the wide range and high magnitude of mitotic
counts observed within feline mammary carcinomas (Fig. 1).
In Mills et al’s15 original publication, the thresholds for the
mitotic counts were 51 and 71 mitoses in 10 high-power
fields according to the observed tertiles in their population. In
the present study, the corresponding thresholds for the mitotic
counts were 34 and 67 to take into account the differences
in field diameter (0.53 mm in Mills et al’s15 study, 0.625 mm in
the present study). Furthermore, the threshold of 33 mitoses
in 10 high-power fields was also evaluated, according to the
receiver-operating characteristic (ROC) curve for 2-year over-
all survival of the cats included in the present study.

REE: Revised Elston and Ellis Histologic Grading System

In the revised Elston and Ellis (REE) grading system (Table 1)
proposed by Mills et al,15 the EE grading system was modi-
fied further to include nuclear form instead of nuclear
pleomorphism.
The nuclear form assessment was evaluated according to
Mills et al15 using a high-power (40) objective in the least
differentiated and/or most invasive portion of the carcinoma.
Subjective evaluation of nuclear shape independent of other
nuclear features or artifactual changes was performed. Devia-
tions from a smooth nuclear contour and oval/round shape such
as corrugation, angularity, clefting (indentation), or overtly
ameboid shape were considered abnormal. 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. Subgroups of 5% (Fig. 2), 6% to 25%, or >25%
(Fig. 3) abnormal were then assigned.
In the REE/LVI scoring system, lymphovascular invasion
was also taken into account, with an additional point when
present.

Figures 1–3. Invasive mammary carcinoma, mammary gland, cat.

Mills-2015 Histologic Grading System Hematoxylin, eosin and saffron (HES). Figure 1. A highly mitotic
Finally, the grading system proposed by Mills et al15 that carcinoma. Figure 2. A carcinoma with low nuclear form score:
included lymphovascular invasion, nuclear form, and mitotic 5% abnormal nuclei. Figure 3. A carcinoma with high nuclear form
count was evaluated (Mills-2015). In the present study, the score: >25% abnormally shaped nuclei. Some nuclei show indentation
(thin arrows), clefting (thick arrow), angularity (black arrowheads), or
Mills-2015 grading system was analyzed using a mitotic count ameboid shape (empty arrowheads).
cutoff of 33 mitoses in 10 high-power fields, according to a
ROC curve for the 2-year overall survival rate of the cats Using this Mills-2015 grading system (Table 2), the absence
included in the present cohort. The chosen cutoff was different of lymphovascular invasion together with less than or equal to
from the >62 cutoff originally proposed by Mills et al,15 which 5% abnormal nuclear form and a mitotic count less than or
corresponded to the median mitotic count observed in their equal to 33 mitoses in 10 high-power fields corresponded to
cohort (by comparison, the median mitotic count in the present grade I (low-grade carcinoma). The presence of any 1 of lym-
cohort was 44 mitoses in 10 high-power fields). phovascular invasion, greater than 5% abnormal nuclear form,
Dagher et al 5

Table 2. Mills-2015 Grading System for Evaluation of Invasive Censored cases in overall survival analyses (ie, in which the
Mammary Carcinoma in Female Cats. studied event was not observed) corresponded to animals alive
Histologic Featurea
at the end of the follow-up period, which in this study was not
shorter than 730 days (2 years). Uncensored cases in specific
Lymphovascular Invasion survival analyses corresponded to animals that died from can-
Absent 0 cer during the follow-up period. Censored cases in specific
Present 1 survival analyses corresponded to animals alive at the end of
Nuclear formb
the follow-up period, animals that died from a cause unrelated
5% abnormal 0
>5% abnormal 1 to cancer, and animals that died from unknown cause.
Mitotic countc
33 mitoses in 10 high-power 0 Statistical Analyses
fields
>33 1 Statistical analyses were conducted using the MedCalc statis-
Total Score Grade (according to Mills tical software (Ostend, Belgium). The Kaplan-Meier method
et al15) and log-rank tests were used to assess overall survival and
0 I (low-grade carcinoma) cancer-specific survival in univariate survival analyses, and
1 II (intermediate-grade carcinoma)
Cox proportional hazards models were used for multivariate
2–3 III (high-grade carcinoma)
Total Score Grade (in the present cohort) survival analyses. The results are reported using the hazard
0 Low-grade FMC ratio (HR), its 95% confidence interval (95% CI), and the P
1–2–3 High-grade FMC value of each covariate. A P value equal or less than .05 was
considered significant. The data analyzed in this study are
Abbreviation: FMC, feline mammary carcinomas.
a
Each feature is evaluated, and scores are assigned and summed. Absence of
available from the corresponding author on reasonable request.
lymphovascular invasion, abnormal nuclear form 5%, and a mitotic count 34
correspond to grade I according to Mills et al15 or low-grade FMC in our
cohort (total score ¼ 0). The presence of any 1 of lymphovascular invasion, Results
abnormal nuclear form >5%, or a mitotic count >34 indicates grade II according
to Mills et al15 or high-grade FMC in our cohort (total score ¼ 1). If any 2 or all Epidemiologic Data
3 features are present, grade III is assigned (total score ¼ 2–3) according to
The cohort comprised 342 female cats with stages I through
Mills et al15 or high-grade FMC in our cohort (total score ¼ 2–3).
b
Abnormal nuclear form includes any deviation from smooth nuclear contour III invasive feline mammary carcinomas. The mean age at
or round/oval nuclear shape such as clefting, angularity, corrugation, or diagnosis was 11.2 + 2.7 years (median ¼ 11.1, range, 4.0–
ameboid morphology assessed at high power (40–60 objective) in the least 21.3 years). The cats were mainly European (297/342,
differentiated and/or most invasive portions of the tumor. The number of
nuclei exhibiting the abnormal nuclear form is estimated and expressed as a
86.8%), 34 were pure-breed cats, including Siamese (n ¼
percentage of the total number of nuclei within any given field. 17), Persians (n ¼ 6), Chartreux (n ¼ 4), Birman (n ¼ 3),
c
Cumulative number of mitoses in 10 consecutive fields in the most mitotically Bombay, British Short Hair, Norwegian, and Oriental (n ¼ 1
active area with a microscope field diameter of 0.625 mm (40 objective). each). Eleven cats were mixed-bred, including 5 cross-
Siamese and 4 cross-Persian cats.
Almost half of the cohort (163/342, 48%) were spayed (by
or a cumulative mitotic count greater than 33 yielded grade II ovariectomy or ovariohysterectomy) before the diagnosis of
(intermediate-grade carcinoma). Finally, if any 2 or all 3 of the mammary carcinoma, and slightly more than half of the cats
aforementioned features were present, grade III (high-grade were intact females (179/342, 52%). Among intact female cats,
carcinoma) was assigned. Afterward, grade II and III tumors it should be noted that 59 cats were spayed at the time of
were grouped together for survival analyses. diagnosis of mammary carcinoma. Very few cats were steri-
lized early, before the age of 2 years (15/342, 4%). More than
one-third of the cats (120/342, 35%) were not spayed all.
Survival Analysis
All cases were followed up for at least 2 years with meticulous
Clinicopathologic Data
emphasis on locoregional relapse (local recurrence and/or The cohort comprised 293 of 342 (85.7%) single invasive
lymph node metastasis) and distant metastasis. Overall survival mammary carcinomas and 49 of 342 (14.3%) multiple (within
was defined as the time period between mastectomy and death a given mammary gland) and multicentric (affecting more than
from any cause, including death attributable to the mammary 1 mammary gland) invasive mammary carcinomas. The histo-
carcinoma. Specific survival was defined as the time period logical types encountered in decreasing order of frequency
between mastectomy and death attributable to the mammary were cribriform (171/342, 50.0%), solid (78/342, 22.8%), tubu-
carcinoma. Uncensored cases in overall survival analyses cor- lopapillary (56/342, 16.3%), and mucinous (37/342, 10.8%).
responded to animals that died during the follow-up period (ie, The mean pathologic tumor size was 17.9 + 7.4 mm, with a
uncensored cases are those in which the studied event, death median of 17.0 mm and a range of 3.0 to 48.0 mm. Lympho-
from any cause, occurred during the follow-up period). vascular invasion was confirmed in 168 of 342 cases (49.1%),
6 Veterinary Pathology XX(X)

central necrosis in 307 of 342 cases (89.7%), squamous differ- associated with a worse overall survival than grade II feline
entiation in 157 of 342 cases (45.9%), and moderate to severe mammary carcinomas (HR ¼ 1.00, reference), whereas there
tumor-associated lymphohistiocytic and plasmacytic inflam- was no significant difference in overall survival between
mation in 176 of 342 cases (51.4%). the 10 cats with grade I carcinomas and the 172 cats with
grade II carcinomas. The EE grading system was not signif-
icantly associated with specific survival (P ¼ .0752, Cox
Histological Grading Systems proportional hazards model).
According to the EE grading system, the cohort comprised 10 The MMEE grading system was significantly associated
of 342 (3.0%) grade I feline invasive mammary carcinomas, with overall survival (P ¼ .0044), MMEE-grade III carcinomas
172 of 342 (50.2%) grade II cases, and 160 of 342 (46.8%) were significantly (P ¼ .0062; HR ¼ 1.66; 95% CI, 1.16–2.38)
grade III cases. In the MMEE grading system, the cohort com- associated with poorer overall survival than MMEE-grade I
prised 75 of 342 (21.9%) MMEE-grade I cases, 204 of 342 carcinomas, whereas MMEE-grade II carcinomas (P ¼
(59.7%) MMEE-grade II cases, and 63 of 342 (18.4%) .0034; HR ¼ 1.55; 95% CI, 1.16–2.08) were significantly dif-
MMEE-grade III cases. In the REE system, there were 104 ferent from MMEE-grade I carcinomas with respect to overall
of 342 (30.4%) REE-grade I carcinomas, 196 of 342 (57.3%) survival (Fig. 5). The MMEE grading system was also signif-
REE-grade II carcinomas, and 42 of 342 (12.3%) REE-grade icantly associated with specific survival (P ¼ .0408); MMEE-
III carcinomas. In the REE/LVI grading system, there were 69 grade III carcinomas were significantly (P ¼ .0309; HR ¼
of 342 (20.2%) REE/LVI-grade I cases, 179 of 342 (52.3%) 1.59; 95% CI, 1.05–2.41) associated with poorer specific sur-
REE/LVI-grade II cases, and 94 of 342 (27.5%) REE/LVI- vival than MMEE-grade I carcinomas, and MMEE-grade II
grade III cases. Finally, based on the grading system proposed carcinomas (P ¼ .0265; HR ¼ 1.47; 95% CI, 1.05–2.06) were
by Mills et al,15 which takes into account lymphovascular inva- significantly different from MMEE-grade I carcinomas with
sion, the nuclear form at threshold >5% abnormal, and the respect to specific survival.
mitotic count at threshold >33 mitoses in 10 high-power fields By multivariate survival analysis, according to the MMEE
(Table 2), there were 15 of 342 (4.4%) Mills-2015 grade I grading system, MMEE-grade III carcinomas (P ¼ .0466,
carcinomas, 78 of 342 (22.8%) Mills-2015 grade II carcinomas, HR ¼ 1.46, 95% CI, 1.01–2.11), MMEE-grade II carcinomas
and 249 of 342 (72.8%) Mills-2015 grade III carcinomas. (P ¼ .0329, HR ¼ 1.39, 95% CI, 1.03–1.88), and MMEE-grade
I carcinomas (HR ¼ 1.00, reference) with decreasing hazard
ratios were significantly associated with a worse overall sur-
Survival Analyses vival, independently from the pathologic tumor size (pT 20
Results of Kaplan-Meier survival analyses and log-rank tests mm: P ¼ .0021, HR ¼ 1.45, 95% CI, 1.15–1.83) and positive
regarding overall and cancer-specific survival according to nodal stage (P ¼ .0012, HR ¼ 1.51, 95% CI, 1.18–1.94). By
patient age at diagnosis, spaying status, pathologic tumor size, multivariate analysis regarding cancer-specific survival,
lymph node metastasis, WHO stage, histologic type, lympho- MMEE-grade III carcinomas (P ¼ .0303, HR ¼ 1.59, 95%
vascular invasion, tumor-associated inflammation, central CI, 1.05–2.41), MMEE-grade II carcinomas (P ¼ .0220,
necrosis, squamous differentiation, tubule formation, nuclear HR ¼ 1.49, 95% CI, 1.06–2.08), and MMEE-grade I carcino-
pleomorphism, nuclear form, and mitotic count are listed in mas (HR ¼ 1.00, reference) with decreasing hazard ratios were
Table 3. significantly associated with a worse specific survival, inde-
Regarding overall survival, a larger pathologic tumor size pendently from a positive nodal stage (P < .0001, HR ¼ 1.82,
(20 mm, HR ¼ 1.61; P < .0001), the presence of lymphovas- 95% CI, 1.38–2.41).
cular invasion (HR ¼ 1.89; P < .0001), lymph node metastasis The REE grading system was first analyzed without includ-
(pNþ, HR ¼ 1.57; P ¼ .0002), a more advanced WHO stage ing lymphovascular invasion, and the results were significantly
(P < .0001), a moderate to severe tumor-associated inflamma- associated with overall survival (P ¼ .0364), but the 42 REE-
tion (HR ¼ 1.33; P ¼ .0125), and a higher mitotic count (cutoff grade III FMCs did not significantly differ from the 104 REE-
value of 33, HR ¼ 1.27; P ¼ .05 being almost significant) were grade I FMCs (P ¼ .2577); there was a significantly worse
all associated with a worse overall survival (Table 3). Regard- overall survival for the 196 REE-grade II FMCs compared to
ing cancer-specific survival, a larger pathologic tumor size REE-grade I FMCs (P ¼ .0114, HR ¼ 1.40, 95% CI, 1.08–
(20 mm, HR ¼ 1.72; P < .0001), the presence of lymphovas- 1.80). The REE grading system without including lymphovas-
cular invasion (HR ¼ 2.17; P < .0001), lymph node metastasis cular invasion was not significantly associated with specific
(pNþ, HR ¼ 1.78; P < .0001), a more advanced WHO stage survival (P ¼ .0746, Cox proportional-hazards model).
(P < .0001), and a moderate to severe tumor-associated inflam- The REE grading system that included lymphovascular inva-
mation (HR ¼ 1.43; P ¼ .0078) were all associated with a sion (REE/LVI) was significantly associated with overall survival
worse specific survival (Table 3). (P ¼ .0005), with its 3 grading categories (I, II, and III) being of
The EE grading system was significantly associated with increasing risk for all-cause mortality (Fig. 6). Compared to REE/
overall survival (P ¼ .0194, Cox proportional-hazards model, LVI-grade I mammary carcinomas considered as the reference
Fig. 4). More precisely, the 160 histological grade III carci- category (HR¼1.00), REE/LVI-grade II carcinomas (HR ¼ 1.56;
nomas (HR ¼ 1.37; 95% CI, 1.09–1.73) were significantly 95% CI, 1.14–2.11; P ¼ .0050) and REE/LVI-grade III
Dagher et al 7

Table 3. Univariate Survival Analysis of Selected Clinicopathologic Criteria With Respect to Overall Survival and Specific Survival in Female
Cats With Invasive Mammary Carcinoma.

Overall Survival Specific Survival

Median Overall
Clinicopathologic Data (N) Survival Time P Valuea HR 95% CI P Valuea HR 95% CI

Age .19 .29

11 years (N ¼ 158) 365 days (12.0 months) .34 0.89 0.71–1.12 .87 0.98 0.75 –1.28
>11 years (N ¼ 169) 332 days (10.9 months) — 1 Referenceb — 1 Referenceb
Spaying status .0056 .04
Intact throughout life (N ¼ 120) 280 days (9.2 months) .01 1.39 1.08–1.78 .01 1.46 1.09 –1.96
Spayed before 2 years (N ¼ 15) 368 days (12.1 months) .34 0.73 0.38–1.38 .84 0.93 0.47 –1.85
Spayed after 2 years, before FMC diagnosis 373 days (12.3 months) — 1 Referenceb — 1 Referenceb
(N ¼ 148)
Spayed at diagnosis of FMC (N ¼ 59) 377 days (12.4 months) .25 0.83 0.60–1.15 .74 0.94 0.64–1.37
Pathologic tumor size <.0001 <.0001
0–19 mm (N ¼ 176) 444 days (14.6 months) 1 Referenceb 1 Referenceb
20 mm (N ¼ 163) 234 days (7.7 months) 1.61 1.28–2.03 1.72 1.32–2.25
Lymph node metastasis .0002 <.0001
Yes (pNþ, N ¼ 97) 234 days (7.7 months) 1.57 1.20–2.06 1.78 1.30–2.44
No (pN0, N¼26) or unknown (pNX, N ¼ 219) 405 days (13.3 months) 1 Referenceb 1 Referenceb
WHO stage <.0001 <.0001
Stage I (N ¼ 137) 495 days (16.2 months) — 1 Referenceb — 1 Referenceb
Stage II (N ¼ 72) 360 days (11.8 months) .0063 1.53 1.13–2.06 .03 1.48 1.03–2.14
Stage III (N ¼ 127) 227 days (7.4 months) <.0001 1.86 1.43–2.40 <.0001 2.2 1.63–2.96
Histological type .16 .36
Cribriform (N ¼ 171) 366 days (12.0 months) — 1 Referenceb — 1 Referenceb
Mucinous (N ¼ 37) 367 days (12.0 months) .25 1.24 0.86–1.81 .34 1.23 0.81–1.88
Solid (N ¼ 78) 272 days (8.9 months) .03 1.37 1.03–1.81 .11 1.3 0.93–1.80
Tubulopapillary (N ¼ 56) 332 days (10.9 months) .82 1.04 0.75–1.44 .85 0.96 0.65–1.42
Lymphovascular invasion
Yes (N ¼ 168) 212 days (7.0 months) — 1 Referenceb — 1 Referenceb
No (N ¼ 174) 480 days (15.8 months) <.0001 0.53 0.42–0.67 <.0001 0.46 0.36–0.61
Inflammation
Moderate to severe (N ¼ 176) 259 days (8.5 months) — 1 Referenceb — 1 Referenceb
Absent to mild (N ¼ 166) 400 days (13.2 months) .0125 0.75 0.60–0.94 .0078 0.7 0.54–0.91
Central necrosis
Yes (N ¼ 307) 344 days (11.3 months) — 1 Referenceb — 1 Referenceb
No (N ¼ 35) 398 days (13.1 months) .58 1.11 0.76–1.61 .47 1.16 0.75–1.79
Squamous differentiation
Yes (N ¼ 157) 292 days (9.6 months) .16 1.18 0.94–1.47 .84 1.02 0.79–1.33
No (N ¼ 185) 367 days (12.1 months) — 1 Referenceb — 1 Referenceb
Tubule formation .15 .31
<10% (N ¼ 52) 454 days (14.9 months) — 1 Referenceb — 1 Referenceb
10–75% (N ¼ 225) 344 days (11.3 months) .07 1.37 0.98–1.92 .24 1.26 0.86–1.85
>75% (N ¼ 65) 354 days (11.6 months) .1 1.39 0.94–2.08 .14 1.41 0.90–2.21
Nuclear pleomorphism .003 .05
1 (N ¼ 6) 564 days (18.5 months) .84 0.91 0.37–2.22 .91 0.94 0.35–2.57
2 (N ¼ 171) 435 days (14.3 months) — 1 Referenceb — 1 Referenceb
3 (N ¼ 165) 249 days (5.4 months) .0008 1.48 1.18–1.86 .02 1.38 1.06–1.80
Nuclear form .21 .6
<5% abnormal (N ¼ 78) 377 days (12.3 months) — 1 Referenceb — 1 Referenceb
5%–25% abnormal (N ¼ 143) 342 days (11.2 months) .31 1.17 0.87–1.57 .85 1.03 0.74–1.44
>25% abnormal (N ¼ 121) 322 days (10.6 months) .07 1.32 0.97–1.79 .37 1.17 0.83–1.66
Mitotic count
33 in 10 HPFs (N ¼ 116) 400 days (13.1 months) .05 0.79 0.63–0.99 .13 0.81 0.61– 1.06
> 33 (N ¼ 226) 332 days (10.9 months) — 1 referenceb — 1 referenceb
Abbreviations: FMC, feline mammary carcinomas; HPF, high-power field; HR, hazard ratio.
a
For clinicopathologic data with at least 3 subgroups, the global P value indicated on top is the P value for the model, indicating whether (or not) the given
clinicopathologic data are significantly associated with overall survival or cancer-specific survival. The other P values indicate whether (or not) a given subgroup is
associated with a significantly different survival probability from the reference subgroup. The reference subgroup is given a hazard ratio of 1.00 and is not
associated with a P value (symbol “—” in the column “P Value”).
b
The reference subgroup is given a hazard ratio of 1.00; by comparison, other subgroups are associated with a worse overall or specific survival if their hazard ratio
is higher than 1.00 (more events, ie deaths, were observed), and their associated P value is less than .05; a subgroup with a HR < 1.00 and P < .05 is associated with a
better overall or specific survival than the reference subgroup.
8 Veterinary Pathology XX(X)

Figures 4–7. Kaplan-Meier curves depicting overall survival of female cats with invasive mammary carcinomas. Figure 4. Prognostic value of
the Elston and Ellis grading system. The 160 EE grade III carcinomas were significantly associated with a worse overall survival than the 172 EE
grade II feline mammary carcinomas. Figure 5. Prognostic value of the mitotic-modified Elston and Ellis grading system. MMEE-grade II and
MMEE grade III carcinomas were significantly associated with poorer overall survival than MMEE-grade I carcinomas. Figure 6. Prognostic value
of the revised Elston and Ellis grading system, which takes lymphovascular invasion into account. The overall survival probability gradually
decreased from REE/LVI-grade I mammary carcinomas to REE/LVI-grade III carcinomas. Figure 7. Prognostic value of the Mills-2015 grading
system with adapted mitotic count cutoff value of 33 mitoses in 10 high-power fields. The 249 Mills-2015 grade III feline mammary carcinomas
(FMCs) were associated with shorter overall survival than the 15 Mills-2015 grade I FMCs. The difference between Mills-2015 grade II and grade
I FMCs was not statistically significant.

carcinomas (HR ¼ 1.93; 95% CI, 1.38–2.71; P ¼ .0002) were P ¼ .0052, HR ¼ 1.43, 95% CI, 1.11–1.83). Regarding cancer-
associated with a significantly poorer overall survival. The specific survival by multivariate analysis, REE/LVI-grade III
REE grading system that included lymphovascular invasion carcinomas (P ¼ .0262, HR ¼ 1.59, 95% CI, 1.06–2.40), REE/
(REE/LVI) was also significantly associated with specific sur- LVI-grade II carcinomas (P ¼ .0505, HR ¼ 1.44, 95% CI,
vival (P ¼ .0018). Compared to REE/LVI-grade I mammary 1.00–2.09), and REE/LVI-grade I carcinomas (HR ¼ 1.00,
carcinomas considered as the reference category (HR ¼ 1.00), reference) with decreasing hazard ratios were associated with
REE/LVI-grade II carcinomas (HR ¼ 1.60; 95% CI, 1.11–2.30; a worse specific survival, independently from the pathologic
P ¼ .0118) and REE/LVI-grade III carcinomas (HR ¼ 2.02; tumor size (pT 20 mm: P ¼ .0014, HR ¼ 1.55, 95% CI, 1.19–
95% CI, 1.35–3.00; P ¼ .0006) were associated with a signif- 2.02) and a positive nodal status (pNþ, P ¼ .0009, HR ¼ 1.61,
icantly greater risk of cancer-related death. 95% CI, 1.22–2.14).
By multivariate analysis regarding overall survival, accord- The Mills-2015 grading system was significantly associated
ing to the REE/LVI grading system, REE/LVI-grade III with overall survival (P < .0001; Cox proportional hazards
carcinomas (P ¼ .0099, HR ¼ 1.58, 95% CI, 1.12–2.24), regression). The 249 Mills-2015 grade III FMCs were associ-
REE/LVI-grade II carcinomas (P ¼ .0285, HR ¼ 1.42, 95% ated with shorter overall survival (HR ¼ 2.94; 95% CI, 1.51–
CI, 1.04–1.93), and REE/LVI-grade I carcinomas (HR ¼ 1.00, 5.73; P ¼ .0017) compared to the 15 Mills-2015 grade I FMCs
reference) with decreasing hazard ratios were significantly (HR ¼ 1.00, reference); however, the 78 Mills-2015 grade II
associated with a worse overall survival, independently FMCs did not significantly differ in overall survival (HR ¼
from the pathologic tumor size (pT 20 mm: P ¼ .0010, HR 1.94; 95% CI, 0.96–3.91; P ¼ .0643) from Mills-2015 grade
¼ 1.47, 95% CI, 1.17–1.85) and a positive nodal status (pNþ, I FMCs (Fig. 7). The Mills-2015 grading system at 33
Dagher et al 9

mitoses cutoff was also significantly associated with specific not significant, as previously reported in feline mammary
survival (P < .0001; Cox proportional hazards regression). The carcinomas.2,11,14,23 However, grade I carcinomas were rare
249 Mills-2015 grade III FMCs were associated with shorter (10/342) and underrepresented when compared with grade II
specific survival (HR ¼ 2.28; 95% CI, 1.35–3.83; P ¼ .0020) (172/342) and grade III (160/342) carcinomas. The low fre-
compared to the 15 Mills-2015 grade I FMCs (HR ¼ 1.00, quency of grade I FMCs was also reported by Mills et al15 and
reference); however, the 78 Mills-2015 grade II FMCs did not Soares et al26 and might be the consequence of the high mitotic
significantly differ in specific survival (HR ¼ 1.15; 95% CI, counts of feline mammary carcinomas compared to human
0.68–1.96; P ¼ .6049) from Mills-2015 grade I FMCs. breast cancers. In Mills et al’s15 study, only 2 of 108 (1.9%)
Afterward, Mills-2015 grade II and III tumors were merged FMCs were grade I, and the classic EE grading system was not
together, and the results were significantly associated with significantly associated with cancer-specific survival.15
overall survival (univariate survival analysis, P ¼ .0031; The classic EE grading system has been previously used to
HR ¼ 2.43; 95% CI, 1.59–3.71 for high-grade FMCs compared grade FMCs. Castagnaro et al2 and Millanta et al14 associated
to low-grade FMCs) and specific survival (univariate survival the histological grade with postsurgery survival of queens with
analysis, P ¼ .0040; HR ¼ 2.76; 95% CI, 1.22–6.20 for high- mammary carcinomas in univariate analysis but failed to
grade FMCs compared to low-grade FMCs). demonstrate an independent prognostic value of the histologi-
By multivariate survival analysis, the high-grade carcino- cal grade by multivariate analysis. Seixas et al23 demonstrated
mas (grades II and III according to Mills-2015 grading system by multivariate analysis that the histologic grade defined
with adapted mitotic count cutoff value of 33) were signifi- according to EE was an independent prognostic factor related
cantly associated with a worse overall survival (P ¼ .0139, to overall survival, with the presence of nodal metastasis as the
HR ¼ 2.33, 95% CI, 1.19–4.54), independently from the patho- independent covariate; but again, only a minority of the carci-
logic tumor size (pT 20 mm, P ¼ .0008, HR ¼ 1.48, 95% CI, nomas were grade I (5/92, 5.4%). These findings warrant a
1.18–1.86) and a positive nodal status (pNþ, P ¼ .0018, HR ¼ more adapted and compatible grading system for feline inva-
1.49, 95% CI, 1.16–1.91). Also, the high-grade carcinomas sive mammary carcinomas.
(grades II and III according to Mills-2015 grading system with When analyzed separately, 2 of the histological criteria used
adapted mitotic count cutoff value of 33) were significantly to define the histological grade had or almost had prognostic
associated with a worse specific survival (P ¼ .0364, HR ¼ significance in the present cohort: the mitotic count (with a
2.40, 95% CI, 1.06–5.43), independently from the pathologic cutoff value of 33 mitoses in 10 400 fields) was almost sig-
tumor size (pT 20 mm, P ¼ .0012, HR ¼ 1.55, 95% CI, 1.19– nificantly associated with overall survival by univariate analy-
2.03) and a positive nodal status (pNþ, P ¼ .0003, HR ¼ 1.68, sis (P ¼ .050), and nuclear pleomorphism was significantly
95% CI, 1.27–2.23). associated with overall survival (P ¼ .003), similar to findings
in previous studies,23 including that of Mills et al15 with respect
to cancer-specific survival. In our study, the nuclear pleo-
Discussion morphism scores showed a significant correlation with overall
Few veterinary studies have attempted to validate previously survival for the second and third subcategories (5%–25% and
published tumor grading schemes in an independent cohort. 25% abnormal nuclei, respectively) in univariate survival
The aim of our study was to evaluate and if possible validate analysis, whereas nuclear form was not significantly associated
the prognostic value of different histologic grading systems, with overall survival. In the study performed by Mills et al,15
including the EE grading system designed for human breast nuclear pleomorphism and nuclear form scores were both
cancers,5 and the modified and newly designed histologic grad- found to be associated with specific survival in univariate sur-
ing systems published lately by Mills et al15 for feline invasive vival analysis. There is thus a rationale for improving the def-
mammary carcinomas.15 In this purpose, we studied 342 stages initions of the mitotic count scores and nuclear pleomorphism/
I through III invasive mammary carcinomas in female cats (the nuclear form scores in feline mammary carcinomas. Three new
largest retrospective FMC cohort described so far). grading systems were proposed by Mills et al15 in an attempt to
The histologic grade of invasive mammary carcinomas is improve the histologic grading of feline mammary carcinoma.
defined minimally by 3 histologic parameters: tubule forma- First, the MMEE grading system adapts the range of mitotic
tion, nuclear pleomorphism, and mitotic count, although there count subcategories to better accommodate the high median
exists variations in the criteria used to quantify these histolo- and broad range of mitotic counts encountered within feline
gical features. In human breast pathology for instance, the invasive mammary carcinomas, as described by Mills et al.15
original Bloom and Richardson1 grading system was modified This modification improves the detection of the previously
by Elston and Ellis5 to improve consistency and reproducibil- underrepresented grade I carcinomas in the classic EE grading
ity.5 In feline mammary carcinomas, there was a need to revise system. In the present study, we confirm that an adaptation of
the classically used criteria defined by Elston and Ellis, notably the mitotic count threshold for feline mammary carcinomas has
because very few cases fall into the grade I subgroup.15,30,31 prognostic value with respect to overall and cancer-specific
In this study, the classic EE grading system5 was applied, survival. Of note, the calculated threshold for the mitotic index
and although it was significantly associated with overall sur- in our cohort (33 mitoses in 10 high-power fields) differs
vival, the difference between grade I and grade II FMCs was from the threshold published by Mills et al15 (>62 mitoses in
10 Veterinary Pathology XX(X)

10 high-power fields). The >62 cutoff corresponds to the med- robustness of lymphovascular invasion as a prognostic factor,
ian mitotic count in Mills et al’s15 cohort. In the present study, but lymphovascular invasion is a feature of the pathologic stage
the median mitotic count was 44 mitoses in 10 high-power of invasive mammary carcinomas rather than a feature of his-
fields; the 33 mitoses cutoff was calculated by ROC curve tological grade of malignancy.
analysis according to the 2-year survival rates of female cats Lymphovascular invasion, the presence of neoplastic emboli
included in the present study. This suggests that the thresholds within lymph and/or blood vessels, is one of the strongest prog-
used for the mitotic index in feline mammary carcinomas prob- nostic factors in female cats with invasive mammary carcino-
ably need in-house validation in a given laboratory for veter- mas.15,19,21,23,28,29 In this study, in case of doubt on HES slides,
inary pathology before they can be used in prognostic purposes. lymphovascular invasion was confirmed (or not) using immuno-
Then, the REE grading system proposed by Mills et al15 was histochemistry to the LMO2 (LIM domain-only protein-2) tran-
evaluated in our cohort. In this design, nuclear pleomorphism scription factor, which gives a nuclear signal in lymphatic
was replaced by a score for nuclear form in an attempt to endothelial cells, whereas in Mills et al’s15 study, lymphovascu-
reduce subjectivity,15 although we felt that nuclear form is in lar invasion was assessed by immunohistochemistry to von Will-
practice as subjective as nuclear polymorphism to evaluate. In ebrand factor. Indeed, lymphovascular invasion may be difficult
this REE grading system proposed by Mills et al,15 lympho- to objectify, either because it may resemble technical retraction
vascular invasion was also added. In our cohort, the REE artifacts on paraffin sections or the lymphatic vessels may be
system was associated with overall survival in univariate completely obstructed by carcinoma cells. In our opinion, the
analysis, and the REE/LVI grading system was significantly immunolabeling of lymphatic endothelial cells is very helpful
associated with overall and cancer-specific survival in both for lymphovascular invasion confirmation and represents a help-
univariate and multivariate analyses, independently from ful prognostic tool, especially when the regional lymph node was
increased pathologic tumor size and positive nodal stage. Of not sampled, to assess whether metastatic spread by the lympha-
note, the REE/LVI grading system was associated with over- tic route was in process.
all survival although 1 of its main parameters (nuclear form) In conclusion, the classic EE grading system, originally
was not significantly associated with overall survival designed for human breast cancers but commonly used in cats,
(P ¼ .21). This is probably mainly due to robustness of lym- can be adapted to feline invasive mammary carcinomas, espe-
phovascular invasion as a prognostic factor in feline mam- cially regarding the 3 points attributed to the mitotic count. Its
mary carcinomas in general19,21,23,28 and in our cohort more application in our retrospective cohort showed a significant
specifically. Although valuable prognostically, lymphovascu- association with overall survival, although grade I carcinomas
lar invasion represents a main weakness of this grading are underrepresented in cats. Lymphovascular invasion is a
system because it refers to the histological stage of feline robust prognostic factor in feline mammary carcinomas, but its
mammary carcinomas (ie, the extent of cancer within the use as a prognostic factor should be independent from any
host) rather than to the histological grade (which is a reflec- grading system. Adapting the number of mitoses might be the
tion of cancer cell differentiation, proliferation, and atypia). best strategy to better categorize the highly proliferative mam-
A novel grading system was also developed by Mills et al15 mary carcinomas encountered in the feline species, so the
and included lymphovascular invasion, mitotic count, and MMEE grading system seems to be a good way to improve
nuclear form. As explained in the methods, mitotic count was routine diagnostic evaluation and prognostication of female
adapted to our cohort with a cutoff value of 33 in contrast to 62 cats with invasive mammary carcinoma.
mitoses in 10 high-power fields in the study of Mills et al.15
Using the Mills-2015 grading system and after gathering grade Acknowledgements
II and III carcinomas for analysis, the Mills-2015 grading sys-
We deeply acknowledge Dr. Anne Patsouris (medical oncologist, ICO
tem proved to be associated with overall and cancer-specific
Integrated Center for Oncology, Angers, France) and Dr. Catherine
survival in univariate and multivariate analyses, independently Ibisch (veterinary oncologist, AMaROC, Oniris, Nantes, France) for
from the pathologic tumor size, nodal stage, and distant metas- their help in reviewing the manuscript. The authors thank Dr. Mélanie
tasis. In their study, Mills et al15 also observed, by univariate Pohu, Dr. Floriane Morio, and Dr. Clotilde de Brito, who helped in
survival analysis, the association of their grading system with collecting the clinical and follow-up data. The authors also thank the
specific survival. However, with this Mills-2015 grading sys- veterinary pathologists (Dr. Jean-Loı̈c Le Net, Dr. Virginie Théau, Dr.
tem, grade I carcinomas were underrepresented in our cohort Pierre Lagourette, Dr. Olivier Albaric, and Dr. Sophie Labrut) who
(n ¼ 15/342, 4.4%), whereas they constituted 20.3% (22/108) performed the initial diagnoses of feline mammary carcinomas as well
of Mills et al’s15 cohort. A low frequency of grade I FMCs as the technicians in histopathology (Mr. Bernard Fernandez, Mrs.
according to Mills-2015 grading system (n ¼ 5/61, 8.2%) was Florence Lézin, and Mrs. Catherine Guéreaud), who made the slides.
also reported by Soares et al.26 Although we found that the Finally, we thank the referring veterinarians and the owners of the cats
included in this study, who gave us the clinical and follow-up data.
Mills-2015 grading system was significantly associated with
overall and cancer-specific survival, the nuclear form, which
is part of its definition, was not of prognostic value in our Declaration of Conflicting Interests
retrospective study. Thus, again, the strong prognostic value The author(s) declared no potential conflicts of interest with respect to
of the Mills-2015 grading system is possibly due to the the research, authorship, and/or publication of this article.
Dagher et al 11

Funding 14. Millanta F, Lazzeri G, Mazzei M, et al. Mib-1 labeling index in feline dysplastic
and neoplastic mammary lesions and its relationship with postsurgical prog-
The authors disclosed receipt of the following financial support for the
nosis. Vet Pathol. 2002;39(1):120–126.
research, authorship, and/or publication of this article: This work was
15. Mills SW, Musil KM, Davies JL, et al. Prognostic value of histologic grading
supported by the French National Cancer Institute (INCa, Institut
for feline mammary carcinoma: a retrospective survival analysis. Vet Pathol.
National du Cancer) with a grant for translational research (INCa- 2015;52(2):238–249.
DHOS 2010, Pr. M. Campone); by a grant for PhD students from the 16. Misdorp W, Weijer K. Animal model of human disease: breast cancer. Am J
Ministry of Education and Higher Education of Lebanon (Dr. Elie Pathol. 1980;98(2):573–576.
Dagher); and by Roche Diagnostics GmbH, Germany, which provided 17. Morris J. Mammary tumors in the cat: size matters, so early intervention saves
financial and technical support for the immunohistochemical charac- lives. J Feline Med Surg. 2013;15(5):391–400.
terization of the carcinomas. The funders had no role in study design, 18. Owen LN, World Health Organization. Veterinary Public Health Unit & WHO
data collection, analysis and interpretation, decision to publish, or Collaborating Center for Comparative Oncology. 1980. TNM Classification of
preparation of the manuscript. Tumours in Domestic Animals/ edited by L.N. Owen. Geneva: World Health
Organization. http://apps.who.int/iris/handle/10665/68618. Accessed April 18,
ORCID iD 2019.
Frédérique Nguyen https://orcid.org/0000-0001-7203-2982 19. Preziosi R, Sarli G, Benazzi C, et al. Multiparametric survival analysis of
histological stage and proliferative activity in feline mammary carcinomas.
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