Prognostic variables in canine multicentric

lymphosarcoma

This paper presents the results of a prospectivestudy to investigate others 1981, Cotter 1983, Carter and
others 1987, Postorino and others 1989,
the prognostic value of clinical staging, histologicalgrading, Hahn and others 1992, Keller and others
immunophenotype, mitotic count and average numbers of 1993, Dobson and Gorman 1994). How-
ever, the response of an individual dog
argyrophilic nucleolar organiser region counts in dogs with multicentric to treatment and duration of remission
lymphosarcoma treated with a standard chemotherapy protocol remain unpredictable.
There have been many attempts to
comprisingvincristine, cyclophosphamideand prednisolone. Forty-nine identify factors that predict therapeutic
dogs were treated according to the study protocol. Univariate and response in canine lymphosarcoma. Few
factors have correlated consistently with
multivariate analysis with regression modellingwas used to evaluate treatment response in different studies;
the prognostic importance of patient and tumour variables upon however, there is increasing evidence that
clinical stage (Squire and others 1973,
tumour response and relapsefree survival. Thirtyseven dogs (76 per Crow 1982, Carter and others 1987,
cent) achieved a complete remission, seven (14 per cent) a partial Keller and others 1993, Dobson and
Gorman 1994, Teske and others 1994a),
remissionand five (10 per cent) failed to respond to treatment. histological grading, immunophenotype
None of the variables examined had a statistically significant effect (Teske and others 1994a) and, more
recently, argyrophilic nucleolar organiser
upon tumour response. Tumour immunophenotypewas the only
region (AgNOR) counts (Vail and others
parameter found to have a significant influence on patient survival, 1996, Kiupel and others 1998) are of
prognostic value. Further studies are still
the hazard ratio for Tcell versus &cell immunophenotypewas 3-99
required to validate these findings and
with 95 per cent confidence interval from 1.399 to 11.372, P 0-035. define the role of such factors as predictors
of prognosis.
This prospective study was primarily
J. M. DOBSON, L. B. BLACKWOOD,
INTRODUCTION designed to examine the prognostic value
E. F. MCINNES, D. E. BOSTOCK,
of mitotic index and &NOR counts in
P. NICHOLLS,T. M. HOATHER
Lymphosarcoma is a commonly occurring, canine multicentric lymphosarcoma, but
AND B. D. M TOM*
spontaneousneoplasm of the dog, account- other variables (histological classification,
Journal of Small Animal Practice (2001) ing for approximately 8.5 to 9 per cent of immunophenotype and clinical details)
42,377-384 all canine tumours (Priester and McKay were included because of their possible
1980) and with a reported annual inci- influence upon the prognosis based on
dence rate of at least 24 to 33 per 100,000 previous clinical studies.
dogs (Dorn and others 1967, Teske 1993).
The multicentric form of the disease is
most common in the dog, in which it MATERIALS AND METHODS
Queen’s Veterinary School Hospital, accounts for 84 per cent of all cases of
University of Cambridge, Madingley lymphosarcoma (Madewell and Theilen Recruitment of dogs
Road, Cambridge CB3 OES
1987). Between January 1995 and March 1996,
*Centre for Applied Medical
Statistics, University of Cambridge, The clinical presentation and clinico- dogs with multicentric lymphosarcoma
Institute of Public Health, University pathological features of canine multicentric were recruited to the study following sub-
Forvie Site, Robinson Way, lymphosarcoma have been well docu- mission of lymph node biopsy material to
Cambridge CB2 2SR
mented (Rosenthal 1982, Couto 1985, the University of Cambridge. Following
L. B. Blackwood’s current address
is University of Glasgow Veterinary Dobson and Gorman 1993). A variety of a diagnosis of lymphosarcoma and the
School, Bearsden Road, Bearsden, chemotherapeutic protocols may be effec- owner’s agreement, basic clinical data were
Glasgow G611QH tive in inducing remission of the disease in requested from the veterinary surgeon,
E. F. Mclnnes’s current address is approximately 80 per cent of dl cases and including haematological examination
Department of Pathology, Papworth
Hospital, Papworth Everard, in producing a significant prolongation (red cell, platelet, total white blood cell
Cambridge CB3 8RE of life (Madewell 1975, MacEwen and and differential counts) and biochemical

JOURNAL OF SMALL ANIMAL PRACTICE VOL 42 AUGUST 2001 377

-
evaluation results (hepatic and renal para-
meters, plasma calcium, total protein and
albumin concentrations), and results of
diagnostic imaging, such as thoracic and
abdominal radiography, plus, in some
cases, ultrasonographic evaluation of liver
and spleen. Evaluation of bone marrow (by
aspirate) was requested where indicated by
cytopenia, lymphocytosis or circulating
abnormal lymphoid cells.
This information was used to stage each
case according to the World Health Orga-
nization Clinical Staging System (Owen
1980). Upon receipt of this clinical data,
the dog was entered into the study and a
treatment protocol was issued to the local
veterinary surgeon. Repeat protocols were
issued at eight-week intervals, following
discussion with the veterinarian responsi-
ble for each dog. Dogs which had been
treated with corticosteroids before diagno-
sis were not included in the study.
Chemotherapy
The treatment regimen used was a standard
low dose ‘COP’ protocol, comprising:
0 ‘C‘ Cyclophosphamide (Endoxana;
ASTA Media): 50 mg/m2 orally every 48
hours or, in small dogs, the equivalent
thereof achieved by dosing with one 50 mg
tablet every three or more days;
0 ‘0’Vincristine (formerly Oncovin):
0.5 mg/m2 intravenouslyevery seven days;
0 ‘P’ Prednisolone: 40 mg/m2 orally daily
for the first seven days, then 20 mg/m2
every 48 hours.
This protocol was selected for ease of
administration, low toxicity and cost
rather than efficacy, in order to optimise
patient recruitment and compliance.
If the disease was in complete remission
after eight weeks of continuous induction
therapy, treatment was reduced to alternate
weeks (ie, one week of treatment followed
by one week off treatment). This was then
reduced to one week in three at 32 weeks
and one week in four at 78 weeks, provid-
ing the animal remained in remission.
Haematological evaluation, to check for
drug-induced myelosuppression, was
recommended every four weeks and, if
378
-
neutropenia (mature neutrophil count of
less than 3-0 x 109/litre) was detected,
cyclophosphamide therapy was stopped
until the neutrophil count recovered. Uri-
nalysis was performed every two to three
weeks to check for proteinuria or haema-
turia as indicators of cyclophosphamide-
induced haemorrhagic cystitis. If
haematuria or clinical signs of cystitis
were detected, cyclophosphamide was
withdrawn and replaced with melphalan
(Alkeran; Glaxo Wellcome) at a dose of
5 mg/m2 orally every 48 hours.
End points
The response to induction therapy was
reported by the attending veterinarian
according to hidher assessment of periph-
eral lymph nodes. Tumour response from
week 4 of treatment was categorised as:
complete remission (CR) - a reduction in
the lymph nodes to normal or subnormal
size; partial remission (PR) - a greater than
50 per cent reduction in lymph node size;
no response (NR) - if the lymph nodes
failed to reduce in size by more than 50 per
cent or if the disease progressed. For those
animals in which turnours attained a partial
or complete remission, the second end
point of the study was the time to
first relapse (ie, on detection of recurrent or
increasing lymphadenopathy).As a measure
of disease-free survival time, the ‘relapse-
free interval’ (RFI) was calculated from
week 4 of induction treatment to the date
of first relapse. Following relapse, some ani-
mals received rescue therapy, but, as this
was not standardised, a comparison of
overall survival times was not undertaken.
Table 1. Kiel classification of nomHodgkin’s lymphoma. Modified from Teske and
others (1994b)
Low grade malignancy
Lyrnphocytic (CLL, MF, Sezary)
Lyrnphoplasrnacytic,
lyrnphoplasrnacytoid
Plasrnacytic
Centrocytic
Centroblastic/centrocytic
(follicular/diffuse)
CLL Chronic lymphocytic leukaemia, MF Mycosis fungoides
JOURNAL OF SMALL ANIMAL PRACTICE
Histochemicaland
immunohktochemicalstudies
Parafin wax-embedded blocks of forma-
lin-fured lymph node biopsy tissue were
used for all the following studies.
Histopathological classification and
mitotic index
Paraffin wax sections, 3 to 4 pm in thick-
ness, were cut, mounted on glass slides,
stained with haematoxylin and eosin using
standard histological techniques and
Examined by light microscopy. The neo-
plastic cells were classified morphologically
using a modification of the Kiel classifica-
tion (Table 1). Some sections contained a
mixed population of tumour cells. Where
one cell type could be identified as being
predominant, the tumour was classified as
such; where this was not the case, the
tumour was classified as ‘mixed’.
In the same sections, the number of
mitotic figures was counted over 10 consec-
utive high power fields (X 500 magnifica-
tion). Only those cells unequivocally in
mitosis were included; cells showing nuclear
degeneration or pyknotic change were
disregarded. Counts were averaged to give
the mean number of mitotic figures per
high power field.
&NOR staining and counting
Four micrometre thick sections of lymph
node biopsy material were dewaxed through
xylene and rehydrated through serial alco-
hols before being washed twice in distilled
water. Sections were stained in the dark for
45 minutes with a silver colloidal solution
and then counterstained with haematoxy-
High grade malignancy
Centroblastic
Lyrnphoblastic (Burkitt‘s type,
convoluted cell type)
lmrnunoblastic
Unclassified
VOL 42 AUGUST 2001
 Table 3. Chemotherapy for multicentric lymphosarcoma:
categorical variables as prognostic factors
Variable Number Frequency Valid per cent
of valid
cases

Gender 49
Male 26 53.1
Female 23 46.9
Neuter status 49
Entire 37 75.5
Neutered 12 24.5
Clinical stage 47
I - -
II 3 6.4
111 32 68.1
Table 2. Chemotherapy for multicentric lymphosarcoma: IV 10 21.3
continuous variables as prognostic factors V 2 4.3
Clinical substage 47
Variable Numberof Mean Median Range Standard a 23 48.9
valid cases deviation b 24 51.1
Cell type 48
Age (years) 49 8.35 8.0 2.0-13.0 2.71 Centroblastic 20 41.7
lmmunoblastic 12 25
Weight (kg) 47 31.61 30.0 8.0-63 14 Lyrnphoblastic 15 31.3
Mitotic count 48 3.2 3.1 0-8.9 2.14 Mixed 1 2.1
lmmunohistochemistry 46
(per HPF)
B cell 38* 82.6
&NOR count 48 6.75 6.55 4.9-11.96 2.5 T cell 6 13
(mean number Null 2 4.3
of AgNORs per cell)
*One tumour classified as B cell also showed T-cell positivity. with an estimated 30 per
HPF High power field cent h e l l and 20 per cent T-cell phenotype

lin, as previously described (Crocker and
Nar 1987). Reactive and normal lymph
node sectionswere used as controls.
The total number of individual
AgNORs in 100 cells was counted
under high power ( x 1000 magnification),
according to the method previously
described by Bostock and others (1989).
AgNOR counts are presented as the mean
value per 100 cells for each tumour.
Immunohistochemistry
Parafin wax-embedded tissue sections, of
3 pm in thickness, were deparafinised in
xylene and rinsed in ethanol. Endogenous
peroxidase activity was blocked by
immersing sections in a 2 per cent solution
of hydrogen peroxide in methanol for
30 minutes. Antigenic sites were
unmasked by microwaving on full power
(800 W) for 10 minutes in an EDTN
citrate buffer solution (Vector, Peterbor-
ough), allowed to cool for 15 minutes and
rinsed in phosphate-buffered saline.
Background staining was reduced with
a casein blocking solution (Biogenex).Pri-
mary antibodies (CD3, 1:lOO dilution and
CD79a, 1:25 dilution, Dako, Ely) were
then applied and incubated in a humidified
tray for one hour at room temperature.
The biotidstreptavidin detection system
(Biogenex)was used following the manu-
facturer’s recommendations and peroxi-
dase activity was visualised by incubation
for three to five minutes with DAB
(3,3-diaminobenzidine,Biogenex). Finally,
sections were lightly counterstained with
Mayer’s haematoxylin, dehydrated and
mounted.
Statistical methods
AU statistical analyses were performed using
the SPSS package (version 9). Age, gender,
neuter status, the clinical stage and substage,
tumour cell type, mitotic count, AgNOR
count and immunophenotype were all
examined for possible influence on the end
points: ‘tumour response’ and ‘relapse-free
interval’. For tumour response the prog-
nostic significance of these variables on CR
versus PR and NR was examined by uni-
variate analysis using Fisher’s exact test for
categorical variables and by logistic regres-
sion with forward and backward methods
to obtain a final model. For the survival
analysis, RFI was examined using Cox’s
proportional hazards models and Kaplan-
Meier survival curves. Cases that were still
alive at the time of writing, that died in
CR or which were lost to follow-up were
censored in these survival analyses.
A total of 49 cases were available for
response and survival analysis, most of
which had a complete record of clinical
and histological details, although a small
number of cases with one or more missing
values have also been included in the study.
Case details, including clinical staging
and the results of histological and immuno-
phenotypic assessments, are summarised
in Tables 2 and 3. A total of 18 breeds
were represented, including eight golden
retrievers, six dobermanns, four West
Highland white terriers, four springer
spaniels, three rottweilers and three
Labrador retrievers. The majority of cases
included in the study (68 per cent) were
presented in clinical stage 111; sub-stages
‘a’ and ’b‘ were equally represented - 49
versus 5 1 per cent, respectively.
Tumour response
Thirty-seven dogs (76 per cent) attained a
CR, seven dogs (14 per cent) achieved a
PR and five dogs (10 per cent) were classi-
fied as NR.
None of the variables examined showed
a significant influence upon tumour
response by univariate analysis (Table 4).
Nor were any variables associated with
Table 4. Univariate, Fisher’s exact
test of tumour response
Variable P-value
Gender (M/F) 0.104
Gender (entire/neutered) 0.703
Clinical stage (I-V) 0.259
Clinical substage (a/b) 0.093
Cell type 0.777
lmmunohistochemistry 1.0

JOURNAL OF SMALL ANIMAL PRACTICE VOL 42 AUGUST 2001 379

-
 Table 5. Outcome of COP chemotherapy: patient survival
Current status Number Comment Analysis
of cases

Alive, tumour in CR 4 1465,1134,1200,1107days Censored

Dead, tumour relapse/ 35 Includes five PR and five NR
failure to respond
Dead. tumour in CR 6 Cause of death: Censored
at time of death tracheal collapse,
haemorrhagic diarrhoea,
laryngeal paralysis. facial pain,
gastrointestinal problems,
neutropenia
Dead, cause of death 2 Practice had record o f death Censored*
and turnour status but no details of cause
at time of death unknown
tumour response when examined by Lost t o follow-up 2 Moved house/away from practice Censored
multiple logistic regression. * A sensitivity analysis was performed for these two dogs where they were also entered into the analysis as a worst case scenario (ie. that
they both died as a result of tumour relapse) and thls dld not change the results

Patient survival
Follow-up details are summarised in Table
5. Thirty-five dogs (71 per cent of cases) -
Table 6. Univariate Cox's regressions of factors associated with relapsefree
survival time
either failed to respond to treatment or
Factor Number of Hazard ratio 95 per cent P-value
relapsed following a period of remission. Six cases confidence interval
dogs died or were euthanased in remission,
two were euthanased for reasons which Age 44 0.976 0.862 - 1.106 0,705
Gender ( M v e r s u s F) 44 1.166 0.569 - 2.393 0.675
could not be ascertained from practice N e u t e r status 44 0.645 0.263 - 1,585 0.339
records and two were lost to follow-up. (N v e r s u s E)
Four dogs were still alive at 1107 to 1465 Bodyweight 42 1.010 0,982 - 1,038 0,497
Clinical stage 42
days and were in complete remission at that (Illv s rest) 0.037
time. All the cases that did not achieve the IV v s I l l 0.662 0,246 - 1.785 0.415
study end point were censored in the sur- v v s 111 4.142 0.514 - 33.414 0,182
II v s 111 4,713 1.326 - 16,747 0.017
vival analysis. The overall median RFI was Clinical substage 42 1.571 0,744 - 3,317 0-236
131 days (95 per cent confidence interval ('b' v s 'a')
Cell t y p e 43 0.9411
[CI]: 95 to 167) (see also Figs 1A and B). (all v s centroblastic)
When each variable was tested for influ- I vs c 0.936 0.364 - 2.408 0.891
ence on W I by univariate Cox's regression, LvsC 1.174 0.493 - 2.797 0.717
M vs C 0,678 0.087 - 5.299 0.711
clinical stage and immunophenotype were Mitotic count 43 1.006 0.988 - 1,025 0,502
of significance.Animals in clinical stage I1 &NOR c o u n t 42 0.999 0.997 - 1.004 0.147
lrnrnunophenotype 39 0-029
had significantly shorter survival times than
T&NvsB
those in clinical stage 111 (hazard ratio [HR]: T vs B 4.143 1.453 - 11.812 0.008
4.713,95 per cent CI: 1.326 to 16.747, P = N vs B 1,475 0.1945 - 11.188 0.707
Tumour r e s p o n s e 44 0.714 0.271 - 1.882 0,496
0.0 17) and the T-cell phenotype was asso- (CR v s rest)
ciated with shorter survival times than that
M Male, F Female, N Neutered, E Entire. I Immunoblastic. C Centroblastic. L Lymphoblastlc. M Mixed. N Null, CR Complete
of the B cell (HR 4.143, 95 per cent CI response Factors with Pvalues<O 3 (shown in bold) were selected for entry into the multivariate Cox's regression model
1.45 to 11.81, P = 0.008) (Table 6). From

1.o 1.o
A B
.0 .8
In 2n
-m

c
0
C
E
&
0

.g
ro
n
.6

.4
-me!
&
0
C
L
0
C
.?
0
n
.6

.4
\i
g g
n n
.2 I k .2

0.0 0.0
200 460 600 800 lW0 1200 1400 1 3 0 200 400 600 800 1000 1200 1400

Disease free survival from 4 weeks (days) Disease free survival from 4 weeks (days)
FIG 1. Kaplan-Meler survlval calculatlonfor all cases. (A) Beat case scenario: the two dogs In whlch the cause of death was not known were censored
as havingdled from a non-tumour-related cause wlth their tumour In CR. (B)Wont case scenario: the same two dogs were Included as havlng dled as
a result of the tumour (let at relapse). On both curves the (+) marks a censoredevent

380 JOURNAL OF SMALL ANIMAL PRACTICE VOL 42 AUGUST ZOO1

-
the univariate analysis, using a cut-off of only parameter shown to affect the relapse- vious epidemiological studies (Rosenthal
P < 0.3, the variables clinical stage, c h i d free interval (overall I‘ = 0.035). The HR 1982, Couto 1985, Dobson and Gorman
substage, &NOR count and immuno- for T-cell versus B-cell immunophenotype 1993). Therefore, the dogs entered into the
phenotype were selected for entry into was 3.99, with the 95 per cent confidence present study are likely to be a representative
the multivariate Cox’s regression model. interval from 1.399 to 11-372.This associ- population of animals afTected by canine
Tumour response was also included, as it ation may also be demonstrated by the multicentric lyrnphosarcoma.The response
was thought that initial response might Kaplan-Meier survival calculation (Fig to the COP chemotherapyregimen (75 per
have an influence on patient survival. 2A), which gives a log-rank statistic of 6.7 cent CR rate and 89 per cent overall
When all variables were examined at one degree of freedom, with P = 0.0096 response [CR + PR] rate) is consistent with
together (Table 7), tumour response was for immunophenotype. previous therapeutic studies of canine mul-
the most significant parameter. Overall, ticentric lymphosarcoma (Madewell 1975,
immunophenotype was not significant, MacEwen and others 1981, Cotter 1983,
although (T versus B phenotype) was sig- DISCUSSION Carter and others 1987, Postorino and oth-
nificant. When either a forward or back- ers 1989, Hahn and others 1992, Keller
ward stepwise approach was used to obtain The demographics observed in this investi- and others 1993, Dobson and Gorman
a final model, immunophenotype was the gation are similar to those reported in pre- 1994). The median RFI of 131 days may
seem comparatively short, but for the pur-
Table 7. Multivariate Cox’s proportional hazard regression model poses of analysis a strict definition of ‘RFI’
was used. As the actual time of achieving
Variable Hazard ratio 95 per cent P-value
confidence interval remission is difficult to assess, the authors
chose to calculate RFI from day 28 (week
Clinical stage 0.214 4) of treatment, by which time maximum
IV vs 111 0,402 0,120 - 1.348 0.14
v vs Ill 5.654 0.626 - 51-061 0.123 response should have been achieved in all
II vs Ill 1.012 0.164 - 6,236 0.99 patients. By definition, patients who did
Clinical substage 0,9076 0.372 - 2.212 0.831
AgNOR count 0.999 0.997 - 1.000
not achieve at least a partial remission by
0.121
lrnrnunophenotype 0.115 day 28 (n = 5) score a RFI of 0 days.
T vs B 5,1003 1.01 - 25.977 0-05
N vs B 2.819 0.300 - 26.516 0,365 Clinical parameters
Tumour response 0.225 0.061 - 0,836 0.026
(CR versus PR/NR) Age, weight and gender were not found
to be prognostic factors for response or

A
1.0 - 6

+- +7

+ +;

.2L
..
+
+

0.0 1 0.00
0 200 400 600 800 1000 1200 1400 II 200 460 600 800 ld00 li00 1400 11 10

Disease free survival from 4 weeks (days) Disease free survival from 4 weeks (days)
FIG 2. KaplamMelersurvival calculation by lmmunophenotype (TGell versus h e l l Immunophenotype). (A) Best case scenarlo: log-rank statlstlc 6.7
at one degree of freedom with P 0.0096. (B) Worst case scenarlo: the curves are essentlally unchanged. Solld h e , T-cell tumours; Broken Ilmt, &cell
tumours. (+) marks a censored event

JOURNALOF SMALL ANIMAL PRACTlCE VOL 42 AUGUST 2001 38 1

-
relapse-free interval. This result is consis-
tent with most previous studies (MacEwen
and others 1981, Postorino and others
1989, Greenlee and others 1990, Hahn
and others 1992, Dobson and Gorman
1994, Teske and others 1994a).
The majority of dogs were presented
with either clinical stage 111 or IV disease
(68 per cent and 21 per cent, respectively),
although bone marrow was not evaluated
in every case and some cases staged as I11 or
IV may have actually been stage V. Clinical
stage did not influence tumour response,
but did have a significant effect on survival
time in the univariate analysis, where
dogs with stage I1 disease appeared to have
a significantly shorter RFI than dogs with
stage I11 disease. The validity of this find-
ing is open to question as there were only
three dogs with stage I1 disease, and two
of these had a T-cell immunophenotype
which could be a major confounding
influence on the result.
Some previous studies have shown
better survival or remission duration for
dogs with early disease (stage I1 or 111)
compared with advanced disease (stage IV
or V) (Squire and others 1973, MacEwen
and others 1981, Crow 1982, Carter and
others 1987, Dobson and Gorman 1994,
Teske and others 1994a). However, just
as many studies have been unable to show a
correlationbetween clinical stage and prog-
nosis (Cotter 1983, MacEwen and others
1987, Postorino and others 1989, Greenlee
and others 1990, Rosenberg and others
1991, Hahn and others 1992). These vari-
able results may reflect the inaccuracy of a
staging process based on clinical examina-
tion, radiography and clinical pathology,
which may over- or underestimate the
extent of the disease. In the absence of
histological or cytological confirmation of
organ involvement, attempts at clinical
staging are to some extent inaccurate.
In accordance with the WHO staging
system, cases in the present series were also
subclassified according to the presence (‘b,
51 per cent) or absence (‘a’, 49 per cent) of
systemic signs. This subclassification did
not correlate with tumour response or sur-
382
vival. As with clinical stage, the value of
clinical substage in prognosis has varied
between different studies. Cotter (1983),
Cotter and Goldstein (1987) and Pos-
torino and others (1989) also reported no
association between substage and progno-
sis, while Hahn and others (1992) and
Keller and others (1993) demonstrated
that performance status or substage was a
significant prognostic factor for survival
duration. Where systemic signs are due to
hypercalcaemia of malignancy, the prog-
nosis is reported to be poorer (Weller and
others 1982, Greenlee and others 1990,
Rosenbergand others 1991). Interestingly,
none of the dogs in the present study were
hypercalcaemicon presentation.
Histologicaltype
Many attempts have been made to classify
canine multicentric lymphosarcoma
according to histological criteria, but a
universally acceptable, clinically relevant
classification system for the disease has not
been agreed. In human medicine, there is
considerable diversity in the microscopic
appearance of non-Hodgkin’s lymphoma
(NHL), which is accompanied by a wide
spectrum of clinical behaviour. Therefore,
NHL may be classified into subgroups,
where the pathological appearance can
be correlated with the clinical behaviour
of the disease (Magrath 1990). Canine
lymphosarcoma shows far less histological
diversity, the majority of turnours being
high grade and of diffuse architecture,
but attempts have been made to modify
human classification systems for use in
the dog. The Kiel classification appears to
be the most appropriate system for canine
lymphosarcoma because it puts little
emphasis on architecture and allows
greater subdivision of the high grade
tumours (Greenlee and others 1990, Teske
and others 1994b).
All of the tumours in the current study
were classified as high grade. The absence
of low grade tumours contrasts with previ-
ous studies reporting between 12 and 24
per cent of canine lymphosarcoma as low
grade, according to the Kiel classification
JOURNAL OF SMALL ANIMAL PRACTICE
(Greenlee and others 1990, Hahn and
others 1992, Teske and others 1994a,
Kiupel and others 1998). The histological
grade of malignancy has been shown to be
a prognostic factor for outcome (Teske and
others 1994a).
The lack of association between cell
type and tumour response or survival is
consistent with some previous studies
(Greenlee and others 1990, Teske and
others 1994b), although Hahn and others
(1992) did demonstrate an association
between Kiel cell type and length of first
remission. There is little consistency
between different studies in the morpho-
logical classification of tumour cell type.
For example, the relatively high incidence
of lymphoblastic morphology in the
present study contrasts with some studies
where the lymphoblastic cell type was not
recorded at all (Teske and others 1994a,
Kiupel and others 1998). A geographical
variation in the incidence of different types
of human NHL in various countries is
recognised (Magrath 1990), and it is
possible that this could account for some
variation in canine lymphosarcoma. A
more likely explanation may lie in the
rather subjective nature of morphological
classification, the fact that most canine
turnours have to some extent a mixed
population of cells and the relative lack
of experience in veterinary medicine of
histological classification of these tumours.
lmmunophenotype
The reagents used for immunopheno-
typing lymphosarcoma tissue (CD3 and
CD79a) have been validated for parafin
sections by several authors (Day 1995,
Milner and others 1996). The present
results are consistent with previous reports
that the majority of canine lymphosarco-
mas are of B-cell phenotype (Appelbaum
and others 1984 [78 per cent], Teske and
others 1994a [71 per cent]), as is the
case in human NHL. Immunophenotype
did not show any association with tumour
response, but had a strong influence on
patient survival, with the T-cell phenotype
associated with significantly shorter RFI.
VOL 42 AUGUST 2001
 This finding is also consistent with other
studies (Teske and others 1994a, Vail and
others 1996).
Two cases in the present series failed
to stain with either antibody and one case
(classified as a B-cell lymphosarcoma)
showed positive stainingwith both reagents,
whereas B cells predominated. A low inci-
dence of non-B-/non-T-cell (or null cell)
lymphosarcoma has been reported in pre-
vious studies of canine lymphosarcoma
(Teske and others 1994b) and this phe-
nomenon is recognised in human NHL. In
the present study, the number of turnours
in this category is too small to draw any
conclusions about prognostic significance.
Dual B- and T-cell positivity has been
reported in some previous studies
(Appelbaum and others 1984, Greenlee
and others 1990), though not in others
(Teske and others 1994a, Fournel-Fleury
and others 1997). Another interesting
observation in the present study was that
histological cell type showed no correlation
with immunophenotype; three of the T-cell
tumours were classified as centroblastic, one
as immunoblastic and two as lympho-
blastic. Therefore, as previously shown,
immunophenotype of canine lympho-
sarcoma cannot be predicted by morpho-
logical criteria (Teske and others 1994b).
Proliferationmarkers
Mitotic count and AgNOR count were
used as objective indicators of tumour pro-
liferation in the present study, but neither
was found to be of prognostic significance.
&NOR counts have been shown to be
useful predictors of prognosis in a variety
of human and canine turnours (Crocker
and Nar 1987, Crocker and Egan 1988,
Bostock and others 1989, 1992) and
recent reports have suggested a role for
&NOR counts as prognostic indicators in
canine multicentric lymphosarcoma (Vail
and others 1996, Kiupel and others 1998).
There is some variation in the AgNOR
values reported for canine lymphosarcoma
between these studies and the present
investigation. Vail and others (1996)
examined tumours from 55 dogs and
JOURNAL OF S M A L L ANIMAL PRACTICE VOL 42
reported a median AgNOR count of 3.5,
with a range of 1.63 to 8.2, while Kiupel
and others (1998) report a range of 1-8to
9.7 in 50 dogs, but provide no overall
mean or median figures. The range in the
present study (4.9 to 11.96) is notably
higher. Kiupel and others (1998) were able
to correlate AgNOR count with tumour
grade, the count being significantly lower
in low grade tumours (mean 3.3) than for
those with high grade tumours (5.7). The
absence of low grade turnours in the pre-
sent study might account for the difference
in AgNOR counts and also offer a possible
explanation for the lack of correlation with
response and survival.
The value of proliferation indices as
prognostic indicators in canine lympho-
sarcoma is questionable. Measurement of
proliferating cell nuclear antigen (PCNA)
did not correlate with survival time in two
studies (Vail and others 1996, Kiupel and
others 1998) and a recent study of PCNA
and Ki-67 in 41 cases of canine multi-
centric lymphosarcoma also failed to
demonstrate a correlation between these
parameters and survival time (Phillips and
others 2000). Because most canine lympho-
sarcomas are high grade, proliferation
indices may not be as useful for predicting
prognosis as they are in other malignant
tumours, where there is a wider spectrum
of tumour grade (such as mammary
tumours). Further work is clearly needed
to define the value of AgNOR counts and
other proliferation indices as prognostic
indicators in canine lymphosarcoma.
Concluslons
This study supports previous findings that
tumour immunophenotype is an impor-
tant prognostic indicator in canine multi-
centric lymphosarcomas, with the T-cell
phenotype associated with significantly
shorter survival times. The lack of correla-
tion with other variables does not necessar-
ily mean that these are not of prognostic
significance in this disease, but where those
associations are relatively weak, a larger
number of cases may be required to pro-
vide the statistical power to detect them.
AUGUST 2001
Acknowledgements
The authors wish to thank all the
veterinary surgeons and owners of dogs
who were involved in this study. Laura
Blackwood was partly funded by Petsavers
as Resident in Clinical Oncology at the
time that the study was conducted.
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