Histological grades of · Endpoints: Development of recurrences/ metastases and neoplasia-associated death. Only animals with complete data were included (n=66). Recruitment of animals,

Histological grades of

malignancy and prognosis

in canine mammary tumors.

Veterinary Faculty

Complutense University of Madrid,

Spain

Laura Peña, Alicia Álvarez,

María López de la Banda, Natividad Martínez,

Mª Dolores Pérez-Alenza.

Histological Malignancy Grading in

canine mammary tumors.

Histological diversity of canine mammary tumors

(CMT) makes difficult their diagnosis.

A grading system has been proposed to better

accurate the tumor information provided from the

pathologist to the clinician.

Histological Malignancy Grading

for human breast cancer

Method for human breast cancer: Elston and Ellis’

/Bloom and Richardson’s/ Nottingham method(Elston and Ellis, 1991)

Tubule formation

Nuclear pleomorphism

Mitotic counts

Directed principally at invasive

(simple) adenocarcinomas.

Elston and Ellis’ Histological

Malignancy Grading in canine

mammary tumors (CMT)

Used in several studies on CMT (Peña et al., 1998; Nieto et al., 2000.; Reis et al., 2003;

Matos et al., 2006; Millanta et al., 2006; Gama et al., 2008; Alves et al., 2008; De Oliveira et al., 2009; Gama et al., 2010; Santos et al., 2010)

Prognostic value in CMT? :

- 1998, 2000: Grade related to survival in univariate but

not in multivariate analyses. (Peña et al., 1998; Nieto et al., 2000.)

- 2005: Grade related to survival in univariate analyses.

NO MULTIVARIATE study. (Karayannopoulou et al., 2005)

Prognostic factors should be evaluated using prospective

multivariate analyses, in order to know whether or not a

factor could predict prognosis independently.

How to evaluate myoepithelial areas ?

Histological Malignancy Grading of CMT:

The need of a canine adapted system

CMT are less aggresive than human breast cancers

(only few metastatize). (Withrow, 2001; Clemente et al., 2010 ).




(only few metastatize).

The existence of complex and mixed malignant

tumors. It is essential to use uniform criteria in assessing

these areas.




(only few metastatize). (Withrow., Clemente et al., 2010 ).



these areas.

Specific criteria of malignancy in canine mammary

tumors (Goldschmidt et al., 2011): Nuclear pleomorphism- even in benign

tumors- is frequent.




(only few metastatize). (Withrow., Clemente et al., 2010 ).



these areas.

Specific criteria of malignancy in canine mammary

tumors (Goldschmidt et al., 2011): Nuclear pleomorphism- even in benign

tumors- is frequent.

Histological Malignancy Grading of

CMT by a canine adapted system

In 2000- start to use a modification of the Elston and

Ellis’ method for CMT.

Related to prognosis? According to clinicians (VTH

Madrid) yes => a prognostic study including the grading

system was needed.



Grading system partially published and compared with a

previous system by Misdorp. (Clemente et al. 2010; Goldschmidt, Peña et al., Vet.

Pathol. 48:117-131, 2011).



Grading system partially published and compared with a

previous system by Misdorp. (Clemente et al. 2010; Goldschmidt, Peña et al., Vet.

Pathol. 48:117-131, 2011).

Retrospective study: Better associated to lymph node

metastases at time of diagnosis than Misdorp’s. (Rasotto et al., Vet

Pathol 0nline published on June 13, 2011).

To study the prognostic value of

histological grade in canine mammary

cancer in a prospective clinical study

with two-years follow-up

AIM

Materials and Methods

Prospective study.

Female dogs with malignant CMT (no sarcomas) presented at

Complutense University Veterinary Teaching Hospital (Madrid), clinically

evaluated and surgically treated through 2008. Clinical stages: I, II

(local), III (local advanced), IV (regional). No distant metastasis (V). No

chemotherapy.

Follow-up: 28-38 months. Clinical evaluations every 4-6 months.

Endpoints: Development of recurrences/ metastases and neoplasia-

associated death.

Only animals with complete data were included (n=66).

Recruitment of animals, criteria of inclusion,

and clinical procedures

Recommended Guidelines for the conduct and evaluation

of prognostic studies in Veterinary Oncology. Vet. Pathol .

48: 7-18, 2011


Performed in 2008 using a modification of the WHO’s

classification for CMT (vet. Pathol, Jan, 2011) (Goldschmidt, Peña

et al., Vet. Pathol. 48:117-131, 2011).

Histopathology

Histologic grading of CMT


Tubular formation (1 to 3 points):

– 1= formation of tubules in >75% – 2= formation of tubules in 10-75% (moderate formation of tubular arrangements admixed with areas of

solid growth) – 3= formation of tubules in (<10%) (minimal or no tubule formation) In complex or mixed tumors, tubular formation is evaluated only in epithelial areas In malignant myoepithelioma tubular formation is 2.

Nuclear pleomorphism (1 to 3 points):

– 1= uniform or regular small nucleus, and occasional nucleoli – 2= moderate degree of variation in nuclear size and shape, hypercromatic nucleus, presence of nucleoli

and some of them can be prominent – 3= marked variation in nuclear size, hypercromatic nucleus, often with one or more prominent nucleoli

In complex or mixed tumors, nuclear pleomorphism is evaluated in all the malignant components.

Mitotic rate (1 to 3 points): – 1= 0 to 9 mitotic figures/10 HPF; – 2= 10 to 19 mitotic figures/10 HPF; – 3= 20 or more mitotic figures/10 HPF.

The fields are selected at the periphery or the most mitotically active parts of the sample (not only epithelial

cells).

Modification of Elston and Ellis’ numeric method (1991)

- Grade I= 3-5 points, well differentiated;

- Grade II= 6-7 points, moderately differentiated;

- Grade III= 8-9 points, poorly differentiated.

Histologic grading of CMT


Tubular formation

Nuclear pleomorphism

Mitotic count

GRADE

Statistical study


Epidemiological variables: Age at diagnosis, spayed status, breed,

weight, regularity of estrus, and previous hormonal treatments.

Clinical variables: Number of neoplasms/animal, location, size in cm,

size (categorical), adherence to skin, adherence to underlying tissues, tumor

ulceration, clinical stage, lymph node status confirmed by cytology). Type of

surgery.

IBM SPSS Statistics 19

Statistical study Materials and Methods

Histological variables:

- Histological diagnosis

- Histol. Diagn. 3 cat

(HD3)

1

- In situ carcinoma.

- Simple carcinoma

- Carcinoma arising

- Complex carcinoma

- Mixed type carcinoma

- Ductal carcinoma

- Adenosquamous carcinoma

2

- Solid carcinoma

- Comedocarcinoma.

- Carcinoma and mal. myoepithelioma

- Anaplastic carcinoma

3

Other types

- Grade: I (n=29), II (n= 20), III (n=17).

- Lymph node metastases (surgical specimen, n=40, H&E)

HD3

Statistical study


Follow-up variables:

• Disease-free Survival (DFS)(months). DFS: Time from surgery to the development of recurrences and/or metastases)

• Overall Survival (OS)(months).

OS: Time from surgery to death by neoplasia or end of the follow-up.

Significant level p<0.05

• Recurrences/metastases (rec/met)(no/yes).

Recurrence: developing of a subsequent CMC at the original tumor location.

• Death by tumor (no/yes).

Results and Discussion

Rec/Met

A) Univariate analyses: Rec/Met, Cancer death

Age (9-11 years) (p=0.01)

Spayed status (p= 0.02)

Clinical stage (p=0.001)

Histological type (p=0.005) and HD3

(p<0.001)

Lymph node metastases (histologically

confirmed) (p=0.003)

Grade (p<0.001)

Cancer death (p<0.001)


0

10

20

30

40

50

60

70

80

90

100

Grade I Grade II Grade III

Rec/Met NO

Rec/Met YES

Rec/Met Grade /

(p<0.001)

%

Cancer death

A) Univariate analyses: Rec/Met, Cancer death

Age (9-11 years) (p=0.009)

Breed (large) (p= 0.04)

Clinical stage (regional) (p<0.001)

Histological type (p=0.01) and HD3

(p<0.001)

Lymph node metastases (histologically

confirmed) (p=0.001)

Grade (p<0.001)



Cancer death Grade /

(p<0.001)

%

0

10

20

30

40

50

60

70

80

90

100

Grade I Grade II Grade III

Cancer death NO

Cancer death YES

DFS OS

B) Survival Study

Kaplan-Meier survival curves


DFS OS

Age 9-11 y. ↓DFS

(p=0.007)

9-11 y. ↓OS

(p=0.005)

B) Survival Study



DFS OS

Age 9-11 y. ↓DFS

(p=0.007)

9-11 y. ↓OS

(p=0.005)

Spayed ↓DFS (p= 0.026)

B) Survival Study



DFS OS

Age 9-11 y. ↓DFS

(p=0.007)

9-11 y. ↓OS

(p=0.005)


Breed large breed, ↓OS

(p=0.027)

B) Survival Study



DFS OS

Age 9-11 y. ↓DFS

(p=0.007)

9-11 y. ↓OS

(p=0.005)



(p=0.027)

Clinical stage local/regional

(p<0.001)

local/regional

(p<0.001)

B) Survival Study



DFS OS

Age 9-11 y. ↓DFS

(p=0.007)

9-11 y. ↓OS

(p=0.005)



(p=0.027)


(p<0.001)

local/regional

(p<0.001)

Tumor size >3cm, ↓OS

(p=0.028)

B) Survival Study



DFS OS

Age 9-11 y. ↓DFS

(p=0.007)

9-11 y. ↓OS

(p=0.005)



(p=0.027)


(p<0.001)

local/regional

(p<0.001)


(p=0.028)

HD3 groups1/2 (p<0.001) groups1/2 (p<0.001)

B) Survival Study



DFS OS

Age 9-11 y. ↓DFS

(p=0.007)

9-11 y. ↓OS

(p=0.005)



(p=0.027)


(p<0.001)

local/regional

(p<0.001)


(p=0.028)


Lymph node met.

(H&E)

(p<0.001) (p<0.001)

B) Survival Study



DFS OS

Age 9-11 y. ↓DFS

(p=0.007)

9-11 y. ↓OS

(p=0.005)



(p=0.027)


(p<0.001)

local/regional

(p<0.001)


(p=0.028)


Lymph node met.

(H&E)

(p<0.001) (p<0.001)

Grade (p<0.001) (p<0.001)

B) Survival Study



B) Survival Study



DFS

(p=0.007)

(p= 0.026)

(p<0.001)

(p<0.001)

(p<0.001)

(p<0.001)

B) Survival Study



DFS

(p=0.007)

(p= 0.026)

(p<0.001)

(p<0.001)

(p<0.001)

(p<0.001)

B) Survival Study



DFS

(p<0.001)

B) Survival Study



OS

(p=0.005) (p=0.027)

(p<0.001)

(p=0.028) (p<0.001) (p<0.001)

B) Survival Study



OS

(p=0.005) (p=0.027)

(p<0.001)

(p=0.028) (p<0.001) (p<0.001)

B) Survival Study



OS

(p<0.001)

C) Survival study. Multivariate

analyses (Cox Regression)


To evaluate the influence of sets of variables on dependent follow-up

variables and find the independent predictive variables.

Step Selected variables

DFS 1 Clinical stage p<0.001

2 Grade p=0.01

Clinical stage

3 Grade

Spayed status p=0.06

Clinical Stage

C) Survival study. Multivariate

analyses (Cox Regression)


Step Selected variables

OS 1 Clinical stage p< 0.001

2 Grade p= 0.018

Clinical stage

3 Grade

Clinical stage

Ulceration p= 0.07

4 Grade

Ulceration

“Chuky”, Shitzu, intact female, 12 y.

Local stage, complex carcinoma, grade I.

Picture during follow-up visit two years

after surgery.

OS= 28 months

Besides the histological type, the grade gives accurate

information to clinician about the histological malignancy.

Easy to apply for clinicians.

Limitations:

- Sarcomas

- Carcinosarcomas (mesenchymal component)

Further studies including low frequent histological

types (Group 3, i.e.carcinosarcomas).

Grading of CMT

Conclusions

The histological grading system of canine

mammary tumors has prognostic value in

prospective univariate and multivariate

analyses (independent prognostic factor).

Conclusions

Grading of CMT

Clinicians after reading a mammary gland

histopathological report…..

… such as: “Diagnosis: tubular/solid

complex carcinoma with foci of

adenosquamous transformation in

benign tumor (mixed type)”

Clinicians after reading a mammary gland histopathological

report…..INCLUDING A GRADE

… such as: tubular/solid complex carcinoma with

foci of adenosquamous transformation in benign

tumor (mixed type), GRADE I.

“The pathologist”

¡Muchas

gracias!

Thank you

very much!

Tack så

mycket!

Documents

Histological grades of · Endpoints: Development of recurrences/ metastases and neoplasia-associated death. Only animals with complete data were included (n=66). Recruitment of animals,