Machine Translated by Google

Received: 4 May 2021 Revised: 18 June 2021 Accepted: 8 July 2021

DOI: 10.1111/vco.12752

ORIGINAL ARTICLE

Outcomes and prognostic factors in canine epitheliotropic and

nonepitheliotropic cutaneous T-cell lymphomas

Kazushi Azuma1 | Aki Ohmi1 | Yuko Goto-Koshino1 | Hirotaka Tomiyasu2 |

Koichi Ohno2 | James K. Chambers3 | Kazuyuki Uchida3 | Hiroyuki Namba4 |
Masahiko Nagata5 | Eiji Nagamine6 | Kazumi Nibe7 | Mitsuhiro Irie2,8 |
Hajime Tsujimoto2

1
Veterinary Medical Center, Graduate School
of Agricultural and Life Sciences, The Abstract
University of Tokyo, Bunkyo-ku, Tokyo, Japan Canine cutaneous lymphoma is an uncommon lymphoma in dogs. Most canine cuta-
2
Department of Veterinary Internal Medicine,
neous lymphoma cases have a T-cell origin. Canine cutaneous T-cell lymphoma
Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Bunkyo-ku, (CTCL) is classified into epitheliotropic and nonepitheliotropic cutaneous lympho-
Tokyo, Japan
mas, and each type of lymphoma is subclassified into several histological subtypes.
3
Department of Veterinary Pathology,
Graduate School of Agricultural and Life Limited information is available regarding the prognostic significance of clinical vari-
Sciences, The University of Tokyo, Bunkyo-ku, ables and histopathological subtypes in dogs with CTCL. This retrospective study
Tokyo, Japan
4
aimed to investigate the influence of clinical variables and histopathological sub-
Namba Pathological Diagnostic Laboratory,
Setagaya-ku, Tokyo, Japan types on the prognosis of dogs with CTCL. Forty-six dogs diagnosed with CTCL by
5
Dermatology, ASC, Chofu-shi, Tokyo, Japan histopathological examination were included. Histopathological specimens were
6
IDEXX Laboratories, Koganei-shi, Tokyo, reexamined and classified into CTCL subtypes. The influence of the type of skin
Japan
7 lesion, histopathological subtype, haematological examination results and treatment
Japan Animal Referral Medical Center
Kawasaki, Kawasaki-shi, Kanagawa, Japan response on the overall survival time (OS) was examined. Thirty-one dogs were
8
Shikoku Veterinary Medical Center, Kagawa, diagnosed with epitheliotropic CTCL (mycosis fungoides in 28 dogs; pagetoid
Japan
reticulosis in 3 dogs) and 15 dogs were diagnosed with nonepitheliotropic CTCL
Correspondence (anaplastic large T-cell lymphoma in 6 dogs; peripheral T-cell lymphoma, not other-
Aki Ohmi, Veterinary Medical Center,
Graduate School of Agricultural and Life wise specified, in 9 dogs). The OS of dogs diagnosed with epitheliotropic CTCL
Sciences, The University of Tokyo, 1-1-1 (141 days) was significantly shorter than that of dogs diagnosed with non-
Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Email: a-ohmi@g.ecc.u-tokyo.ac.jp epitheliotropic CTCL (374 days). As clinical variables, the presence of neoplastic
lymphocytes in peripheral blood, thrombocytopenia and initial chemotherapeutic
response was related to prognosis. Our results demonstrated that histopathological
subtype and several clinical variables were found to influence the prognosis of dogs
with CTCL.

KEYWORDS

CCNU, chemotherapy, dog, lomustine, mycosis fungoides, World Health Organization

1 | INTRODUCTION dogs at risk.1–4 Although lymphoma accounts for 7%–24% of all canine
neoplasia and 83% of all canine haematopoietic malignancies,5,6 the
Lymphoma is one of the most common neoplasms in dogs, and its cutaneous variant is relatively rare and accounts for approximately 5%
annual incidence is estimated to range from 13 to 114 per 100,000 of all canine lymphomas.7 –10 Several studies

Vet Comp Oncol. 2021;1–9. wileyonlinelibrary.com/journal/vco © 2021 John Wiley & Sons Ltd 1
Machine Translated by Google
2 AZUMA ET AL.
have shown the efficacy of chemotherapeutic agents for the treatment of canine
cutaneous lymphoma and survival time of the
patients.11,12
Canine cutaneous lymphoma mostly originates from T-cells and
canine cutaneous T-cell lymphoma (CTCL) is classified into epi-theliotropic and
nonepitheliotropic lymphomas.9 Furthermore, canine
epitheliotropic CTCL has been subclassified into three subtypes:
mycosis fungoides (MF), pagetoid reticulosis and Sézary syndrome.
Canine nonepitheliotropic CTCL has also been subclassified into three
subtypes: anaplastic large T-cell lymphoma (ALTCL), subcutaneous
panniculitis-like T-cell lymphoma and peripheral T-cell lymphoma, not
otherwise specified (PTCL-NOS).13
In humans, the prognosis of CTCL depends on the histopathologi-cal
subtypes, with 5-year survival rates of MF, pagetoid reticulosis,
Sézary syndrome, ALTCL, subcutaneous panniculitis-like T-cell lym-
phoma and PTCL-NOS reported to be 80%, 100%, 24%, 95%, 82% and
16%, respectively.14,15 MF is the most common CTCL and MF is generally
considered an indolent disease in humans. In contrast, the prognosis of canine
epitheliotropic CTCL is reported to be poor, and the
Median remission duration is generally short (3–7 months).16–19 Some
clinical factors, such as the presence or absence of multiple lesions, the
distribution of the lesions (cutaneous or mucocutaneous/mucosal) and
chemotherapy intervention have been shown to relate to prognosis in
canine epitheliotropic CTCL.10 As for nonepitheliotropic lymphoma,
There have been few reports on the prognosis of the disease. The median
survival time in canine inflamed nonepitheliotropic CTCL, which is a
unique variant of nonepitheliotropic CTCL, was reported to be
9 months.20 No other report has comprehensively investigated the
prognosis of canine nonepitheliotropic CTCL. Furthermore, the difference in
prognosis amongst the histopathological subtypes of canine
CTCL has not yet been reported. In the present study, histopathological
subtype and clinical features including signalment, gross lesion description,
haematological examination results and therapeutic response were
retrospectively surveyed in dogs with epitheliotropic and non-epitheliotropic CTCL,
and the influence of these variables on prognosis
was analyzed.
2 | MATERIALS AND METHODS
2.1 | Canine patients
The medical records of dogs referred to the Veterinary Medical Center of the
University of Tokyo and other private hospitals and histo-
logically diagnosed with CTCL between January 2007 and November
2016 were reviewed. Cases that did not show CD3 positivity on
immunohistochemistry were excluded.
Information obtained from medical records included patient char-
acteristics (breed, age, sex and body weight), World Health Organization (WHO)
clinical substage (a or b), pruritus, lymphadenopathy,
gross appearance of cutaneous and mucosal lesions, results of blood
examination (complete blood count, microscopic observation of
peripheral blood smear and serum biochemical analysis), thoracic
radiography, abdominal ultrasonography, lymph node aspiration cytol-ogy,
therapeutic agents used, response to therapy, overall survival
time (OS) and progression-free survival time (PFS).
The evaluation of clinical substage, pruritus, lymphadenopathy,
gross appearance of cutaneous and mucosal lesions, results of blood
examination, thoracic radiography, abdominal ultrasonography and
cytology were performed at the day on which dogs were referred.
Response to therapy was recorded after each treatment and categorized according
to the response evaluation criteria for solid tumors in
dogs (v1.0) published in a Veterinary Cooperative Oncology Group
consensus document: complete response (CR), disappearance of all
injuries; partial response (PR), at least 30% reduction in the sum of
diameters of measurable lesions; progressive disease (PD), either the
appearance of one or more new lesions or at least a 20% increase in
the sum of diameters of measurable lesions; stable disease (SD), less
than 30% reduction or 20% increase in the sum of diameters of mea-
surable lesions.21 CR, PR or SD was documented for a minimum of
1 weeks while receiving chemotherapy.
2.2 | Histological examination and
immunohistochemistry
Tissue samples were fixed in 10% buffered formalin and embedded in
paraffin. Four micrometer-thick sections were deparaffinized and sta-
ined with haematoxylin and eosin. Histopathological specimens of
canine CTCL were subjected to immunohistochemistry and categorized into
histological subtypes, namely, MF, pagetoid reticulosis,
ALTCL, subcutaneous panniculitis-like T-cell lymphoma and PTCL-NOS.13
Immunohistochemistry was performed using the following pri-mary antibodies:
rabbit polyclonal anti-CD3 antibody (ready to use;
Dako, Glostrup, Denmark) and rabbit polyclonal anti-CD20 antibody
(1:400; Thermo Fisher Scientific, Waltham, MA, USA). despues de
deparaffinization, heat-induced antigen retrieval was performed for
CD3 and CD20 immunohistochemistry by autoclaving the sections
for 10 min at 121C in citrate buffer solution (pH 6.0). The sections
were then treated with 1% hydrogen peroxide in methanol at room
temperature for 15 min and then incubated with 8% skim milk in Tris-
buffered saline at 37C for 40 min to block nonspecific reactions. Sub-
Sequently, the sections were incubated overnight with primary anti-CD3 and anti-
CD20 antibodies. After washing in Tris-buffered saline,
the sections were incubated with horseradish peroxidase-labelled
polymer (Dako EnVision+ System; Dako, Carpinteria, CA, USA). Sec-
tions were washed in Tris-buffered saline, and binding of the antibody
was visualized with 0.05% 3,30 -diaminobenzidine and 0.03% hydrogen
peroxide in Tris/HCl buffer. Finally, the sections were counterstained
with Mayer's haematoxylin. Primary antibodies were omitted to pro-
produces negative controls.
Two pathologists (JKC and KU) reviewed the histopathology samples
retrospectively and histopathological diagnosis was made by con-sensus of these
two pathologists according to the WHO classification
of lymphoid neoplasms in animals.13
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AZUMA ET AL.
23 | Polymerase chain reaction for antigen
receptor gene rearrangement (PARR) analysis of
TCRÿ and IgH
Genomic DNA was extracted from preserved formalin-fixed, paraffin-
embedded lesional tissues using the QIAamp DNA FFPE tissue kit
(QIAGEN, Hilden, Germany) according to the manufacturer's instructions
retrospectively. PARR analysis of TCRÿ and IgH was performed as
previously described.22 Briefly, the PCR mixture was composed of
1 AmpliTaq Gold 360 Master Mix (Applied Biosystems, Foster City,
CA, USA), forward primers labeled with fluorescent dyes, reverse
primers, and 100 ng of DNA template. Cycle conditions consisted of an
initial denaturation and enzyme activation step at 95C for 5 min,
followed by 40 cycles of denaturation at 94C for 15 s, annealing at
62C or 56C for 30 s and extension at 72C for 30 s and a final
extension at 72C for 30 min. Each PCR was performed in duplicate.
One microliter of PCR product diluted 20-fold was combined with
8.5 ÿl of Hi-Di formamide (Applied Biosystems) and 0.5 ÿl of 600 LIZ
Size Standard (Applied Biosystems) and subjected to GeneScan
analysis. Distinct peaks that were at least 2-fold higher than the other
background peaks were defined as clonal.
2.4 | Statistical analysis
OS and PFS were calculated using Kaplan–Meier survival curves. For
OS calculations, dogs were censored if they were still alive or lost to
follow-up. OS was measured from the day on which dogs were newly
referred to veterinary hospitals as having cutaneous or mucosal lesions
diagnosed with CTCL to the date of death. PFS was defined as the
date from which treatment started to the date of PD or death from
any cause. All statistical analyzes were performed using commercial
software packages (JMP Pro, 11.2.0' SAS Institute Inc., Cary, NC, USA).
2.5 | Cell line validation statement
Cell line validation testing has not been conducted, as there were no
cell lines used in this study.
3 | RESULTS
3.1 | Histopathological examination and
immunohistochemistry
Histopathological subclassification of CTCL in 46 dogs led to diagnosis
of epitheliotropic CTCL and nonepitheliotropic CTCL in 31 and
15 of these dogs, respectively.
Of the epitheliotropic CTCL cases, 28 and 3 dogs were diagnosed
with MF and pagetoid reticulosis, respectively. MF was characterized
by infiltration of neoplastic lymphocytes from the dermis to the
3
epidermis and adnexa (Figure 1(A)). Neoplastic lymphocytes had clear
cytoplasm and a medium-sized nucleus [1.5–2 times the size of a red
blood cell (RBC)]. Pagetoid reticulosis was characterized by detach-
ment of the epidermis and infiltration of neoplastic lymphocytes lim-
ited to the epidermis (Figure 1(B)). Neoplastic lymphocytes had scarce
cytoplasm and a small nucleus (1–1.5 times the size of RBC).
In nonepitheliotropic CTCL cases, 6 and 9 dogs were diagnosed
with ALTCL and PTCL-NOS, respectively. ALTCL was characterized
by infiltration of neoplastic lymphocytes in the dermis and/or subcu-
taneous tissue (Figure 1(C)). The neoplastic lymphocytes had indistinct
cellular borders and a large nucleus (2–3 times the size of RBC).
Marked nuclear atypia and anisocytosis were observed. PTCL-NOS
(9 dogs) was characterized by infiltration of neoplastic lymphocytes in
the dermis and/or subcutaneous tissue (Figure 1(D)). The neoplastic
lymphocytes had a small- to medium-sized nucleus (1–2 times the size
of RBC). Infiltration of inflammatory cells, such as eosinophils and
macrophages, was prominent in PTCL-NOS.
The 46 dogs included in the present study had cutaneous lym-
phomas positive for CD3. Of these 46 dogs, 37 were negative for CD20
and 9 dogs were positive for both CD3 and CD20 (MF, 7 dogs; Pagetoid
reticulosis, 1 dog and ALTCL, 1 dog).
3.2 | PARR analysis
Biopsy specimens from 40 dogs with CTCL underwent PARR. Thirty of
the 40 dogs with CTCL showed monoclonal TCRÿ rearrangement.
Two dogs showed clonal rearrangement of both IgH and TCRÿ. In the
other 8 dogs, clonal rearrangement was not detected in either IgH or
TCRÿ by PARR. Clonal rearrangement of TCRÿ was detected in 23 of
26 dogs (88%) with epitheliotropic CTCL and in 9 of 14 dogs (64%) with
nonepitheliotropic CTCL.
3.3 | Clinical characteristics
Of the 46 dogs that underwent histopathological subclassification of
CTCL, 40 dogs with full clinical data were reviewed for prognostic
analyses. The study population included a golden retriever (n = 8),
mixed breeds (n = 5), Chihuahua (n = 3), Maltese (n = 3), miniature
dachshund (n = 3), Yorkshire terrier (n = 3), Pembroke Welsh corgi (n =
3), beagle (n = 2), French bulldog (n = 2) and one each of the popular
breeds: Cavalier King Charles spaniel, Shih Tzu, Shiba dog, toy poodle,
German shepherd dog, Labrador retriever, Shetland sheepdog and
Bernese mountain dog. The median age was 11 (range, 3–14) years
and the median body weight was 10.85 (range, 1.7–39.2) kg.
Eighteen dogs were female (15 neutered, 3 intact) and 22 dogs were
male (8 neutered, 14 intact).
At the initial examination, 32 dogs did not show any systemic clinical
signs categorized as substage a and 8 dogs showed systemic clinical
signs categorized as substage b.
Of the 40 dogs with CTCL for which full medical records were
available, major site of the lesion was observed in the skin (23 dogs)
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4 AZUMA ET AL.

FIGURE 1 Histopathological features of CTCL in dogs in the present study. (A) Mycosis fungoides (haematoxylin and eosin stain, 40; inset, 400).
The neoplastic lymphocytes with clear cytoplasm and a medium-sized nucleus (1.5–2 times the size of RBC) infiltrated into the dermis and
epidermis is observed. The inset shows a Pautrier's microabscess, characterized by infiltration of neoplastic lymphocytes into the epidermis.
(B) Pagetoid reticulosis (haematoxylin and eosin stain, 100). Infiltration of the neoplastic lymphocytes with scarce cytoplasm and a small nucleus (1–
1.5 times the size of RBC) is limited to the epidermis. (C) Anaplastic large T-cell lymphoma (haematoxylin and eosin stain, 40; inset, 1000).
The neoplastic lymphocytes contain a large nucleus (2–3 times the size of RBC) and show marked atypia and anisocytosis. Infiltration of these
cells into the dermis and/or subcutaneous tissue is observed. (D) Peripheral T-cell lymphoma, not otherwise specified (haematoxylin and eosin
stain, 40; inset, 1000). Infiltration of neoplastic lymphocytes with a small- to medium-sized nucleus (1–2 times the size of RBC) have infiltrated into
the dermis and/or subcutaneous tissue along with inflammatory cells such as eosinophils and macrophages is observed. CTCL, cutaneous T-
cell lymphoma; RBC, red blood cell

and mucosae (17 dogs). The types of lesions in the skin were nodules
(n = 17, 74%), erythema (n = 12, 52%), erosion (n = 6, 26%), alopecia
(n = 6, 26%), crusts (n = 5, 22%) and scales (n = 3, 13%). Six dogs
(26%) had solitary cutaneous lesions and 17 dogs (74%) had multiple
cutaneous lesions. The types of lesions in the mucosae were erythema
(n = 9, 53%), swelling (n = 8, 47%), erosion (n = 8, 47%), nodules (n =
6, 26%) and depigmentation (n = 3, 13%). Twelve dogs (71%) had
solitary mucosal lesions and 5 dogs (29%) had multiple mucosal
lesions. Ten of the 40 dogs showed pruritus. Anaemia was observed in
3 of the 40 dogs (median, 33.3%; range, 33.0–34.6%; reference range,
37.3–61.7%). Thrombocytopenia was observed in 5 of the 40 dogs
(median, 91,000/ÿl; range, 30,000–141,000/ÿl; reference range, 148,000–
484,000/ÿl). Hypercalcemia was observed in 1 of the 26 dogs for which
serum biochemical analyzes were available (13.3 mg/dl; reference
range, 9.3–12.1 mg/dl). Peripheral blood
Smears of 32 dogs were microscopically evaluated, and neoplastic
large lymphoid cells were found in 5 dogs, all of which were diagnosed
with MF. Fine needle aspiration of the enlarged peripheral lymph nodes
in 29 dogs revealed an increase in neoplastic large lymphoid cells in 10
dogs, but not in the remaining 19 dogs.
Thoracic radiography revealed neither lymph node enlargement
nor lung involvement in the 37 dogs examined. Abdominal ultra-sound
was performed in 37 dogs; abnormalities were found in the liver
(hepatomegaly, 2 dogs; nodules, 2 dogs; heterogeneous echogenicity,
1 dog), spleen (splenomegaly, 3 dogs; nodules, 3 dogs; heterogeneous
echogenicity, 3 dogs) and intraperitoneal lymph node
(enlargement, 4 dogs). Fine needle aspiration in 1 of the 37 dogs with
a hepatic nodule revealed vacuolar degeneration of hepatocytes.
Cytological evaluation of the abdominal organs was not performed in
the other dogs.
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AZUMA ET AL. 5

3.4 | Chemotherapy n = 23), melphalan (2–4 mg/m2 orally every 24 h) + prednisolone (n =

14), Lomustine (CCNU) + L-asparaginase + prednisolone (LAP-based
Thirty-four dogs received at least one type of chemotherapy. protocol,24 n = 6), chlorambucil ( 2 mg/m2 orally every 24 h) +
Chemotherapeutic protocols included vincristine + cyclophosphamide + prednisolone (n = 2) and prednisolone alone (n = 2). Response rates
doxorubicin + L-asparaginase + prednisolone (L-CHOP-based protocol,23 after treatment with prednisolone alone, chlorambucil + prednisolone,

FIGURE 2 Legend on next page.

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6 AZUMA ET AL.

melphalan + prednisolone, the L-CHOP-based protocol and the LAP-
based protocol were 0%, 0%, 43%, 57% and 67%, respectively. The
mediate PFS in dogs treated with melphalan + prednisolone, the L-
CHOP-based protocol and the LAP-based protocol was 53, 21 and
60 days, respectively.
Regarding first-line chemotherapy in each case, the initial chemo-
therapeutic agents included the L-CHOP-based protocol (n = 16),
melphalan + prednisolone (n = 14), the LAP-based protocol (n = 1),
chlorambucil + prednisolone (n = 1) and prednisolone alone (n = 2).
The number of dogs that showed CR, PR, SD and PD to initial chemo-
therapy were 2, 13, 11 and 8, respectively. The initial overall response
rate (CR + PR) was 44% (15/34), whereas the biological response rate
(CR + PR + SD) was 76% (26/34).
Second-line chemotherapy was initiated when the initial chemo-
therapeutic agents were ineffective. Second-line chemotherapy
included the L-CHOP-based protocol (n = 6), the LAP-based protocol
(n = 4) and chlorambucil + prednisolone (n = 1). Third-line chemo-
therapeutic agents included the L-CHOP-based protocol (n = 1) and
the LAP-based protocol (n = 1).
(Figure 2(B)). The treatment response after initial chemotherapy was
found to influence the prognosis of dogs with CTCL. Dogs which
PD showed had shorter OS than those which showed a biological
response (CR, PR and SD) after initial chemotherapy (Figure 2(C)). The
other clinical variables investigated in this study showed no relationship
with the prognosis (Table 1).
The median OS in dogs diagnosed with epitheliotropic CTCL
and nonepitheliotropic CTCL was 141 (12–1776) and 374 (129–
1538) days, respectively. OS in epitheliotropic CTCL cases was significantly
shorter than that in nonepitheliotropic CTCL cases
(Figure 2(D)). The median OS in each subtype was as follows: MF,
138 (13–1776) days; pagetoid reticulosis, 251 (63–1074) days;
ALTCL, 659.5 (266–1365) days and PTCL-NOS, 353 (129–1538) days
days (Figure 2(E)). The median OS in dogs with CD3+CD20+ cutaneous
lymphoma was 138 (16–266) days and OS in dogs with
CD3+CD20 cutaneous lymphoma was 310 (13–1776) days, indicating the
presence of significant difference between the two
groups (Figure 2(F)).
No relationship between the results of clonal rearrangement evaluation
uated using PARR and prognosis was observed.

3.5 | Prognostic analysis

In total, 35 out of the 46 dogs died during the study period. One of
the dogs were suspected to have developed gastric dilation and vol-vulus.
The other 34 dogs died due to deterioration of the disease and
were considered to be tumor-related deaths. Necropsy was not per-
formed in any of the dogs. None of the dogs were euthanized during
this study. The remaining 11 dogs were censored for the prognostic
analysis; 10 dogs were lost to follow-up and the other 1 dog was alive
at the time of data collection. Median follow-up time in the 11 dogs
censored was 277 days (range, 43–1776 days). The median OS for
dogs that received any chemotherapy was 265 (39–1776) days and
the median PFS was 38 (14–1676) days. The presence of neoplastic
lymphocytes in peripheral blood and thrombocytopenia were significantly
related to OS. The dogs with neoplastic lymphocytes in the
peripheral blood had shorter OS than those without neoplastic lym-
phocytes in the peripheral blood (Figure 2(A)). The dogs with
thrombocytopenia had shorter OS than those without thrombocytopenia
4 | DISCUSSION
Previous studies have shown that the OS of dogs with epitheliotropic
CTCL ranges from a few months to 2 years.11,12,16–19 Because eutha-
nasia is frequently requested in canine cutaneous lymphoma cases in
advanced stages, the current OS in these dogs has been difficult to
determine. Furthermore, there has been only one report describing
the prognostic factors of canine CTCL.10 In the present study, no dog
was euthanized, allowing an accurate analysis of the prognosis of
CTCL itself. As a result, the presence of neoplastic lymphocytes in
peripheral blood, thrombocytopenia, initial chemotherapeutic
response, histopathological subtype and CD20 expression in immuno-
histochemistry were demonstrated to influence the prognosis of
canine CTCL.
The presence of neoplastic lymphocytes in the blood was
observed in 5 dogs diagnosed with MF, which meant that these dogs
Could be categorized as Sézary syndrome. Although there have been a

FIGURE 2 Kaplan–Meier survival curves depicting OS in different patient groups. Dogs lost to follow-up or alive at the completion of the
study were censored (tick marks). (A) Dogs that had neoplastic lymphocytes in the peripheral blood (solid line; median survival, 129 days; n = 5)
and dogs that did not have neoplastic lymphocytes in the peripheral blood (dashed line; median survival, 259 days; n = 27). p = .0488. (B) Dogs
showing thrombocytopenia (solid line, median survival 196 days, n = 5) and dogs that did not show thrombocytopenia (dashed line, median
survival 310 days, n = 27). p = .0263. (C) Dogs showing progressive disease after initial chemotherapy (solid line; median survival, 129 days;
n = 8) and dogs that showed biological response (complete response, partial response and stable disease) after initial chemotherapy (dashed line;
medium survival, 300 days; n = 26). p = .0108. (D) Dogs diagnosed with epitheliotropic CTCL (solid line; median survival, 141 days; n = 31) and
dogs diagnosed with nonepitheliotropic CTCL (dashed line; median survival, 374 days; n = 15). p = .0071. (E) Dogs diagnosed with MF (median
survival, 138 days; n = 28), pagetoid reticulosis (median survival, 251 days; n = 3), ALTCL (median survival, 659.5 days; n = 6) and PTCL-NOS
(median survival, 353 days; n = 9). (F) Dogs that had cutaneous lymphoma showing CD3+CD20+ in immunohistochemistry (solid line; median
survival, 138 days; n = 9) and dogs that had cutaneous lymphoma showing CD3+CD20 in immunohistochemistry (dashed line; median survival,
310 days; n = 37). p = .0031. ALTCL, anaplastic large T-cell lymphoma; CTCL, cutaneous T-cell lymphoma; MF, mycosis fungoides; OS, overall
survival time; PTCL-NOS, peripheral T-cell lymphoma, not otherwise specified
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AZUMA ET AL. 7

TABLE 1 Influence of clinical and histopathological variables on the prognosis of dogs with CTCL

Variable n Median OS (days) HR 95% CI p-value

Clinical substage to 32 310 1 .057

b 8 251 0.407 0.156–1.063

Pruritis Forks 10 279.5 0.725 0.311–1.691 .533

No 30 310 1

Major site of the injury Cutaneous 23 271 0.962 0.473–1.957 .915

Mucosal 17 290 1

Number of the injury Solitaire 17 310 1 .691

Multiple 23 271 0.865 0.422–1.772

mass formation Forks 23 310 0.560 0.271–1.158 .113

No 17 251 1

Lymphadenopathy Forks 26 247.5 0.561 0.254–1.239 .148

No 14 339 1

Anaemia Forks 3 163 0.330 0.096–1.129 .200

No 37 251 1

Presence of neoplastic lymphocytes in peripheral blood Forks 5 129 0.373 0.135–0.928 .0488*

No 27 259 1

Thrombocytopenia Forks 5 196 0.336 0.122–0.926 .0263*

No 35 310 1

Hypercalcemia Forks 1 271 N.D. N.D. N.D.

No 25 251

Initial chemotherapeutic response P.S. 8 129 0.323 0.131–0.797 .0108*

C.R., P.R., S.D. 26 300 1

Clonality in PARR Positive 32 267 0.575 0.218–1.515 .3. 4. 5

Negative 8 485 1

Histopathological subtype Epitheliotropic 31 141 0.372 0.176–0.786 .0071*

Nonepitheliotropic fifteen 374 1

CD20 Positive 9 138 0.254 0.099–0.653 .0031*

Negative 37 310 1

Abbreviations: CI, confidence interval; CTCL, cutaneous T-cell lymphoma; CR, complete response; HR, hazard ratio; ND, not determined; OS, overall
survival; PARR, polymerase chain reaction for antigen receptor gene rearrangement; PD, progressive disease; PR, partial response; SD, stable disease.
*Indicating the presence of significant difference.

small number of reports on Sézary syndrome in dogs and its clear defi-
nition has not been established, the disease is generalized epi-
theliotropic T-cell lymphoma with leukaemia, considered to be a
variant of MF with a poor prognosis.25 In this study, the classification
of Sézary syndrome was intentionally excluded because peripheral
blood smear evaluation was not performed in all dogs. Also, the
presence of neoplastic lymphocytes in the blood is conceivably due to the
infiltration of neoplastic lymphocytes into the bone marrow, although
bone marrow examination was not conducted in any of the dogs.
Therefore, poor prognosis in dogs with neoplastic lymphocytes in
peripheral blood might be due to the advanced stage of the disease.
Thrombocytopenia is possibly due to the infiltration of neoplastic lym-
phocytes into the bone marrow, increased platelet consumption asso-
ciated with thrombosis or destruction by secondary immune-
mediated thrombocytopenia, resulting in a shorter OS in these dogs.
In previous reports on the treatment of canine cutaneous lym-
phoma, CCNU has been often employed.10–12 The median response
duration in dogs with epitheliotropic CTCL treated with CCNU was
94–106 days.11,12 However, in a recent review, CCNU is not
necessarily recommended in terms of long-term prognosis.26 In our present
study, dogs were treated with prednisolone alone, chlorambucil +
prednisolone, melphalan + prednisolone and the L-CHOP-based and
LAP-based protocols. Of these chemotherapeutic agents, the LAP-
based protocol, including CCNU, showed the highest response rate.
However, the response rate with CCNU in this study (67%) was
slightly lower than that in previous studies (78–83%),11,12 conceivably
owing to a small number of dogs treated with CCNU as an initial ther-
apy in this study. Prednisolone alone, chlorambucil + prednisolone,
melphalan + prednisolone and the L-CHOP-based protocol showed
numerically lower response rates, suggesting that these
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8 AZUMA ET AL.
chemotherapeutic agents might be less effective than CCNU for the
treatment of canine CTCL. Although several studies demonstrated
clinical responses to CCNU in dogs with epitheliotropic CTCL, the
response duration was reported to be approximately 3 months.11,12
Further efforts are needed to develop more effective treatment for
canine epitheliotropic CTCL.
In humans, cutaneous lymphoma is subclassified into many his-
tological subtypes, and the therapeutic protocol is dependent on the
histopathological subtype.15 In dogs, because subclassification of
CTCL was recently introduced, the prognosis of each subtype has not
been compared and treatment recommendations for each sub-type
have not been established.13 To the authors' knowledge, this study is
the first to report comprehensively on the prognosis of canine
nonepitheliotropic CTCL. The present study demonstrated
that OS in dogs diagnosed with epitheliotropic CTCL was significantly
shorter than that in dogs diagnosed with nonepitheliotropic CTCL.
Furthermore, dogs diagnosed with MF had a tendency toward shorter
OS than pagetoid reticulosis in epitheliotropic CTCL, and dogs with
ALTCL also showed a tendency toward longer OS than PTCL-NOS in
nonepitheliotropic CTCL, but there was no significant difference in OS
due to a small number of cases in each histopatho-
logical subtype.
In humans, MF is considered to be a slowly progressive indolent
disease in general with a 5-year survival rate of approximately 80%.15
However, the prognosis of MF in dogs is considered to be poor in
general because the duration of response to chemotherapy is not
long.11,12,27 In this study, OS in MF was 138 days, the shortest of all
histological subtypes identified. Furthermore, several studies indicated
that lymphocytes in canine MF mostly expressed CD3, CD8 and TCRÿ
unlike common human MF but might be identical to that in primary
cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma
characterized in the WHO-European Organization for Research and
Treatment of Cancer classification for cutaneous lym-phoma in
humans.15,28,29 Furthermore, the 5-year survival of primary cutaneous
aggressive epidermotropic CD8+ cytotoxic T-cell lym-phoma in humans
was reported to be 32% with a median survival of 12 months.30 These
results suggest that canine MF may not be a counterpart of human MF
but resembles primary cutaneous aggres-sive epidermotropic CD8+
cytotoxic T-cell lymphoma as listed in a revised fourth version of the
WHO classification in humans.31 It is
conceivable that canine MF clearly differs from human MF, and the
establishment of therapeutic guidelines for canine MF is needed apart
from that for human MF.
CD20+ MF has been reported in humans, and some reports
suggest that the prognosis of MF with CD20 expression might be worse
than that of MF without CD20 expression.32–34 In this study, OS in
dogs with CD3+CD20+ cutaneous lymphoma was significantly shorter
than that in dogs with CD3+CD20 cutaneous lymphoma, as shown in
humans. Although the reason for the CD20 positivity in canine cutaneous
lymphoma cells is unclear, it can be hypothesized.
that CD20 is activated in a subset of infiltrating cells following their
malignant conversion and enhanced potential for rapid cell cycle pro-
gression, as indicated in humans.32–34 Thus, CD20 expression is
reflective of the aberrant nature of the T-cell population and might be
related to a worse prognosis in dogs, as in humans.
In this study, clonal rearrangement was not detected in either IgH
or TCRÿ by PARR in 8 dogs. It is conceivable that the fragmentation of
DNA was responsible for the false-negative result because genomic
DNA was extracted from preserved formalin-fixed, paraffin-
embedded lesional tissues. Another possibility is that proliferation of
neoplastic cells could not be detected by the primers used in this
study.
This study has several limitations inherent to its retrospective
nature, including incomplete staging of patients, treatment variation and
evaluation of the chemotherapeutic response by different veterinarians
inarians. Moreover, because several lesions in the skin and mucosae
did not form masses, the treatment response could not be objectively
judged based on the size of the lesions. Immunohistochemical double-
staining for CD3 and CD20 was not conducted in this study.
Although a large proportion of neoplastic lymphocytes were positive for
both CD3 and CD20 in cases with CD3+CD20+ lymphoma,
immunohistochemical double-staining was considered to be needed to
demonstrate the double positivity for CD3 and CD20. The number
ber of cases in this study was small, and it was not possible to com-
pair prognoses and prognostic factors amongst the detailed
histopathological subtypes. Studies using a large number of dogs are
needed to further identify prognostic factors and develop more effective
treatment modalities for the clinically problematic
canine CTCL.
5 | CONCLUSION
OS in dogs diagnosed with epitheliotropic CTCL was significantly shorter
than that in dogs diagnosed with nonepitheliotropic CTCL.
Several clinical variables (presence of neoplastic lymphocytes in
peripheral blood, thrombocytopenia and initial chemotherapeutic
response) and CD20 expression in conjunction with CD3 expression
were associated with poor prognosis of canine CTCL. Further studies
are needed to overcome the poor prognosis of
canine CTCL.
CONFLICT OF INTEREST
The authors declare no conflict of interest. None of the authors of this
article has a financial or personal relationship with other people or
organizations that could inappropriately influence or bias the content of
this paper.
DATA AVAILABILITY STATEMENT
Data available on request from the authors.
ORCID
Aki Ohmi https://orcid.org/0000-0001-7592-6391
Hirotaka Tomiyasu https://orcid.org/0000-0001-7708-6218
Kazuyuki Uchida https://orcid.org/0000-0003-4823-0318
Hajime Tsujimoto https://orcid.org/0000-0002-3771-6235
Machine Translated by Google
AZUMA ET AL.
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How to cite this article: Azuma K, Ohmi A, Goto-Koshino Y,
et al. Outcomes and prognostic factors in canine
epitheliotropic and nonepitheliotropic cutaneous T-cell
lymphomas. Vet Comp Oncol. 2021;1-9. https://doi.org/10.
1111/vco.12752