Comparison of epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) immunohistochemical expression and outcomes in canine nasal carcinomas treated with radiation
therapy
Biopsy samples and clinical data Sixty-seven biopsy samples were collected from dogs diagnosed with nasal carcinomas before treatment at Rakuno Gakuen University Veterinary Teaching Hospital (RGU-VTH) between 2004 and
2013. All tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin. The histopathological diagnoses were made by veterinarians in the Department of Veterinary
Pathology at RGU. Medical records and computed tomography (CT) images at the time of diagnosis were reviewed. Clinical staging was evaluated using CT imaging and a previously described staging system  . Fine-needle aspiration of regional lymph nodes was performed if nodes were enlarged. Immunohistochemistry and protein expression scoring Serial sections were prepared from paraffin-embedded tissues for immunohistochemical (IHC) examinations. Antigen retrieval was performed using proteinase K (20 μg/mL, Dako, Glostrup,
Denmark) or by autoclave using 0.1 M citrate buffer (pH 6.0). IHC was performed using an avidin-biotin-peroxidase complex procedure using a commercial kit (Vectastain ABC Kit, Vector
Laboratories, Burling, CA, USA). Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 10 min. summarizes information on primary antibodies against human EGFR and COX-2, including dilutions, incubation times, and pretreatment conditions for antigen retrieval. Staining
was developed using 3-3′-diaminobenzidine (Kanto Chemical Co., Tokyo, Japan) and counterstained with Mayer’s hematoxylin. Negative controls were incubated with isotype-matched, irrelevant
monoclonal or irrelevant polyclonal IgG instead of the specific primary antibody. EGFR and COX-2 immunostaining was scored to semi-quantitatively estimate the degree of protein expression in tumor tissues, based on a previous report . The IHC score was determined from the percentage of positively labeled tumor cells and their average staining intensity. The score
was assessed at ×200 magnification, and the score was given as an average of five fields of view . The total IHC score was calculated by multiplying the scores for the percentage and intensity to give a total score from 0 to 12. Tumor samples with an IHC score ≥2 were
defined as having positive expression. An IHC score of 4 was defined as the threshold separating tumors with high expression from those with low expression. RT and treatment procedure Dogs were treated with radiation using an orthovoltage X-ray machine (TITAN-450S, GE, Tokyo, Japan) with a filter of 1.0 mm of aluminum, 0.3 mm of copper, and 0.5 mm of tin. The exposure
rate was 1.68 Gy/min. The distance from the X-ray source to the skin was 60 cm. The half-value layer was 4.8 mm copper at 450 kV and 10 mA. A single dorsal portal field was delivered. The
radiation field included the tip line of the nose to the top of the head, which was assessed using CT images. The planning target volume included a 1–2 cm margin of normal tissue. The dogs
were treated with ten fractions of 4 Gy on alternate days (Monday, Wednesday, and Friday) for a total dose of 40 Gy to evaluate the relationship between IHC score and outcomes. Statistical analysis Statistical significance of differences was analyzed using the chi-squared (χ 2 ) test for the relationship between protein expression and clinical variables (sex, age, clinical
stage, and cribriform plate destruction). Dogs were monitored and followed up every three months after RT. Follow-up information was collected from medical records or by contacting the
referring veterinarians. Endpoints were: alive if the dog was known to be alive at the time of data collection; lost to follow-up, if the survival status was unknown at the time of data
collection; or dead if the dog had died before data collection. The progression-free interval (PFI) was defined as the time between the first day of RT and the day of disease progression
(local recurrence, metastatic disease, or death). The overall survival time was calculated from the start day of RT to the time of death or the final follow-up. The correlation between
survival and protein marker (EGFR or COX-2) level was estimated using Kaplan-Meier survival curves, and statistical differences between survival curves were calculated using the log-rank
method. P -values of <0.05 were considered significant. StatMate III commercial software (ATMS Co., Ltd., Tokyo, Japan) was used to perform the statistical analysis. 

Sixty-seven biopsy samples were collected from dogs diagnosed with nasal carcinomas before treatment at Rakuno Gakuen University Veterinary Teaching Hospital (RGU-VTH) between 2004 and
2013. All tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin. The histopathological diagnoses were made by veterinarians in the Department of Veterinary
Pathology at RGU. Medical records and computed tomography (CT) images at the time of diagnosis were reviewed. Clinical staging was evaluated using CT imaging and a previously described staging system  . Fine-needle aspiration of regional lymph nodes was performed if nodes were enlarged. 

Serial sections were prepared from paraffin-embedded tissues for immunohistochemical (IHC) examinations. Antigen retrieval was performed using proteinase K (20 μg/mL, Dako, Glostrup,
Denmark) or by autoclave using 0.1 M citrate buffer (pH 6.0). IHC was performed using an avidin-biotin-peroxidase complex procedure using a commercial kit (Vectastain ABC Kit, Vector
Laboratories, Burling, CA, USA). Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 10 min. summarizes information on primary antibodies against human EGFR and COX-2, including dilutions, incubation times, and pretreatment conditions for antigen retrieval. Staining
was developed using 3-3′-diaminobenzidine (Kanto Chemical Co., Tokyo, Japan) and counterstained with Mayer’s hematoxylin. Negative controls were incubated with isotype-matched, irrelevant
monoclonal or irrelevant polyclonal IgG instead of the specific primary antibody. EGFR and COX-2 immunostaining was scored to semi-quantitatively estimate the degree of protein expression in tumor tissues, based on a previous report . The IHC score was determined from the percentage of positively labeled tumor cells and their average staining intensity. The score
was assessed at ×200 magnification, and the score was given as an average of five fields of view . The total IHC score was calculated by multiplying the scores for the percentage and intensity to give a total score from 0 to 12. Tumor samples with an IHC score ≥2 were
defined as having positive expression. An IHC score of 4 was defined as the threshold separating tumors with high expression from those with low expression. 

Dogs were treated with radiation using an orthovoltage X-ray machine (TITAN-450S, GE, Tokyo, Japan) with a filter of 1.0 mm of aluminum, 0.3 mm of copper, and 0.5 mm of tin. The exposure
rate was 1.68 Gy/min. The distance from the X-ray source to the skin was 60 cm. The half-value layer was 4.8 mm copper at 450 kV and 10 mA. A single dorsal portal field was delivered. The
radiation field included the tip line of the nose to the top of the head, which was assessed using CT images. The planning target volume included a 1–2 cm margin of normal tissue. The dogs
were treated with ten fractions of 4 Gy on alternate days (Monday, Wednesday, and Friday) for a total dose of 40 Gy to evaluate the relationship between IHC score and outcomes. 

Statistical significance of differences was analyzed using the chi-squared (χ 2 ) test for the relationship between protein expression and clinical variables (sex, age, clinical
stage, and cribriform plate destruction). Dogs were monitored and followed up every three months after RT. Follow-up information was collected from medical records or by contacting the
referring veterinarians. Endpoints were: alive if the dog was known to be alive at the time of data collection; lost to follow-up, if the survival status was unknown at the time of data
collection; or dead if the dog had died before data collection. The progression-free interval (PFI) was defined as the time between the first day of RT and the day of disease progression
(local recurrence, metastatic disease, or death). The overall survival time was calculated from the start day of RT to the time of death or the final follow-up. The correlation between
survival and protein marker (EGFR or COX-2) level was estimated using Kaplan-Meier survival curves, and statistical differences between survival curves were calculated using the log-rank
method. P -values of <0.05 were considered significant. StatMate III commercial software (ATMS Co., Ltd., Tokyo, Japan) was used to perform the statistical analysis. 

Biopsy samples and clinical data Of the 67 tissue samples, 29 were ADCs, 17 were CAs, 12 were SCCs, and nine were TCCs . All dogs underwent skull CT scans before biopsy. The CT images at the time of diagnosis were reviewed, and clinical staging was evaluated. Twelve dogs (17.9%) had T1 disease,
five (7.5%) had T2 disease, 17 (25.4%) had T3 disease, and 33 (49.2%) had T4 disease. Fine-needle aspiration of regional lymph nodes was performed on 39 dogs. Five dogs had evidence of
regional lymph node metastasis. EGFR and COX-2 protein expression EGFR staining was predominantly membranous and occasionally cytoplasmic . Fifty-nine samples (88.1%) were positive. Positive EGFR expression was observed in 24 of 29 ADC (82.8%), 14 of 17 CA (82.4%), all 12 SCC (100%), and all nine TCC (100%) samples. The
total IHC score for EGFR expression ranged from 0 to 12. The median total IHC score was 6, and the mean was 6.9, while mean IHC scores for ADC, CA, SCC, and TCC were 5.7, 6.3, 9.9, and 8.2,
respectively. Low EGFR expression was found in 20 samples (11 ADCs, six CAs, one SCC, and two TCCs) and high in 47 samples (18 ADCs, 11 CAs, 11 SCCs, and seven TCCs). COX-2 staining was predominantly cytoplasmic and perinuclear . Fifty-five samples (82.1%) were positive. Positive COX-2 expression was observed in 24 of 29 ADC (82.8%), 13 of 17 CA (76.5%), ten of 12 SCC (83.3%), and eight of nine TCC (88.9%)
samples. The total IHC score for COX-2 expression ranged from 0 to 12. The median total IHC score was 6, and the mean was 5.3, while mean IHC scores for ADC, CA, SCC, and TCC was 4.7, 5.6,
6.1, and 5.7, respectively. Low COX-2 expression was found in 32 samples (16 ADCs, seven CAs, five SCCs, and four TCCs) and high in 35 samples (13 ADCs, ten CAs, seven SCCs, and five
TCCs). Neither expression of EGFR nor COX-2 was strongly associated with T staging, cribriform plate destruction, sex, or age . There was no relationship between the expression of EGFR and COX-2 ( P =0.407). Associations between protein expressions and survivals Thirty-eight dogs, including 18 ADCs, nine CAs, nine SCCs, and two TCCs, completed RT as their only curative treatment with no evidence of metastatic disease. According to medical records,
nineteen dogs did not receive any treatments, three dogs did not complete ten fractions (two received only eight, and one received nine treatments due to skin radiation side effects), and
seven dogs received different radiation protocols (3 or 6 Gy per fractions). Of these 38 dogs who completed RT, seven were staged as T1, two as T2, ten as T3, and 19 as T4. Follow-up
intervals ranged from 40 to 1,979 days, with a median follow-up interval of 220 days. Thirty-one dogs died during the study and follow-up period, and seven dogs were lost to follow-up.
Twenty-one died from tumor progression (fourteen had tumor local progression which was diagnosed by head CT scan, and seven had lymph nodes and/or distant metastatic diseases), two from
euthanasia, three from other diseases, and five from unknown causes. Median PFI and overall survival times (MST) were 150 and 236 days, respectively. Kaplan-Meier and log-rank analysis
revealed no significant correlations between PFIs and protein levels of EGFR or COX-2 expression ( P =0.201 and 0.692, respectively). Dogs with low EGFR had longer survival
times than those with high EGFR (MST: 262 days versus 187 days, respectively; P =0.043) . However, COX-2 expression was not correlated with survival (MST: 236 days versus 237 days, respectively; P =0.635) . 

Of the 67 tissue samples, 29 were ADCs, 17 were CAs, 12 were SCCs, and nine were TCCs . All dogs underwent skull CT scans before biopsy. The CT images at the time of diagnosis were reviewed, and clinical staging was evaluated. Twelve dogs (17.9%) had T1 disease,
five (7.5%) had T2 disease, 17 (25.4%) had T3 disease, and 33 (49.2%) had T4 disease. Fine-needle aspiration of regional lymph nodes was performed on 39 dogs. Five dogs had evidence of
regional lymph node metastasis. 

EGFR staining was predominantly membranous and occasionally cytoplasmic . Fifty-nine samples (88.1%) were positive. Positive EGFR expression was observed in 24 of 29 ADC (82.8%), 14 of 17 CA (82.4%), all 12 SCC (100%), and all nine TCC (100%) samples. The
total IHC score for EGFR expression ranged from 0 to 12. The median total IHC score was 6, and the mean was 6.9, while mean IHC scores for ADC, CA, SCC, and TCC were 5.7, 6.3, 9.9, and 8.2,
respectively. Low EGFR expression was found in 20 samples (11 ADCs, six CAs, one SCC, and two TCCs) and high in 47 samples (18 ADCs, 11 CAs, 11 SCCs, and seven TCCs). COX-2 staining was predominantly cytoplasmic and perinuclear . Fifty-five samples (82.1%) were positive. Positive COX-2 expression was observed in 24 of 29 ADC (82.8%), 13 of 17 CA (76.5%), ten of 12 SCC (83.3%), and eight of nine TCC (88.9%)
samples. The total IHC score for COX-2 expression ranged from 0 to 12. The median total IHC score was 6, and the mean was 5.3, while mean IHC scores for ADC, CA, SCC, and TCC was 4.7, 5.6,
6.1, and 5.7, respectively. Low COX-2 expression was found in 32 samples (16 ADCs, seven CAs, five SCCs, and four TCCs) and high in 35 samples (13 ADCs, ten CAs, seven SCCs, and five
TCCs). Neither expression of EGFR nor COX-2 was strongly associated with T staging, cribriform plate destruction, sex, or age . There was no relationship between the expression of EGFR and COX-2 ( P =0.407). 

Thirty-eight dogs, including 18 ADCs, nine CAs, nine SCCs, and two TCCs, completed RT as their only curative treatment with no evidence of metastatic disease. According to medical records,
nineteen dogs did not receive any treatments, three dogs did not complete ten fractions (two received only eight, and one received nine treatments due to skin radiation side effects), and
seven dogs received different radiation protocols (3 or 6 Gy per fractions). Of these 38 dogs who completed RT, seven were staged as T1, two as T2, ten as T3, and 19 as T4. Follow-up
intervals ranged from 40 to 1,979 days, with a median follow-up interval of 220 days. Thirty-one dogs died during the study and follow-up period, and seven dogs were lost to follow-up.
Twenty-one died from tumor progression (fourteen had tumor local progression which was diagnosed by head CT scan, and seven had lymph nodes and/or distant metastatic diseases), two from
euthanasia, three from other diseases, and five from unknown causes. Median PFI and overall survival times (MST) were 150 and 236 days, respectively. Kaplan-Meier and log-rank analysis
revealed no significant correlations between PFIs and protein levels of EGFR or COX-2 expression ( P =0.201 and 0.692, respectively). Dogs with low EGFR had longer survival
times than those with high EGFR (MST: 262 days versus 187 days, respectively; P =0.043) . However, COX-2 expression was not correlated with survival (MST: 236 days versus 237 days, respectively; P =0.635) . 

Most canine nasal carcinomas demonstrated expression of EGFR (88.1%) and COX-2 (82.1%). Although previous reports investigated EGFR and COX-2 IHC expression in canine nasal carcinomas , one study evaluated the potential correlation between protein markers expression and outcome after RT . Unlike in human
medicine, IHC has not been widely used to evaluate outcomes of RT in veterinary studies. Survivin expression was a negative predictor in canine nasal carcinomas treated with RT . Therefore, our study aimed to calculate correlations between IHC and clinical variables and outcomes in canine nasal carcinomas. EGFR plays a crucial role in regulating the cell cycle, angiogenesis, differentiation, and survival . In
a previous study, 54.2% had positive expression in 24 canine nasal carcinomas . In another report, EGFR immunoreactivity was noted in 14 of 16
(87.5%) samples, and messenger RNA for EGFR was detected in 15 of 16 samples. Phosphorylation of EGFR was demonstrated in 10 of 16 samples . In the
present study, 67 samples were evaluated, and 59 tested positive. EGFR positivity was 82.8% in ADC, 82.4% in CA, 100% in SCC, and 100% in TCC. Overexpression of EGFR has been reported to
correlate with poor survival in some human and veterinary tumors . However, the correlation between EGFR overexpression and outcomes in dogs with nasal tumors has not been studied. Our result found that EGFR
overexpression is associated with shorter overall survival in dogs with nasal carcinoma. Combined treatment using an EGFR inhibitor (cetuximab) and RT was superior to RT treatment alone. RT + cetuximab increases the duration of local control and survival in human head and neck
cancers . Another EGFR inhibitor (gefitinib) inhibited activation of anti-apoptotic and proliferative signal transduction pathways to enhance
radiosensitivity in human lung cancer cells . In the present study, nearly all canine nasal carcinomas overexpressed EGFR; this may be considered a
similar therapeutic target in dogs with nasal carcinoma. Previous studies reported that a high percentage (71–90%) of canine nasal carcinoma expressed COX-2 . Similarly, COX-2 was positive in 82.1% of our 67 samples. COX-2 expression inhibits apoptosis and causes
tumor metastasis and angiogenesis in human NPC ; these phenomena have been associated with outcomes . Few studies reported associations between COX-2 expression and outcomes in dogs with cancers. As in our study,
one study found no association between COX-2 expression and survival in dogs with nasal carcinoma treated with hypofractionated RT . Although the role
of COX-2 in carcinogenesis appears straightforward, the prognostic value of COX-2 expression in canine cancer is not well understood. COX-2 levels measured by IHC might not indicate actual
enzymatic activity . A few studies reported the radiosensitizing effects of COX-2 selective inhibitors in human cancer cells; however, the mechanisms of these effects are poorly understood . Celecoxib (a COX-2 inhibitor) increased radiosensitivity in NPC cells. This result highlighted the relationship between G2-M
phase arrest and apoptosis induction . Celecoxib caused the downregulation of COX-2 and vascular endothelial growth factor in human cervical cancer
cells. COX-2 inhibition and RT may appear to synergistically inhibit tumor proliferation and angiogenesis . In our result, most of our nasal carcinoma samples (82.1%) expressed positive in COX-2. Although no relationship was revealed between COX-2 level and survival in dogs with nasal
carcinoma treated with RT in our and a previous study , the combination effect of RT and COX-2 inhibitor has been studied recently . Cancedda et al. combined treatment of RT and firocoxib for canine nasal carcinomas. Despite the absence of statistical evidence for
survival differences between firocoxib and placebo groups, the quality of life was significantly improved by combining firocoxib and RT . Currently, most of veterinary radiation oncology facility are using megavoltage radiation machine. Megavoltage radiation has greater penetrability and dose distribution and would be expected
to be superior to orthovoltage RT for nasal tumors in dogs . This phenomenon is particularly evident in tumors located at greater depths from the
skin surface. Orthovoltage radiation was used in recent study because of the limitations of RT units in our institution. We observed median PFI and MST of 5 and 7.9 months, respectively, which
is similar to the outcomes described in previous canine nasal tumor studies involving orthovoltage treatment . Computer-based conformal RT planning techniques, including three-dimensional conformal (3DCRT) and intensity-modulated radiation therapy (IMRT), are designed to maximize the tumor
dose while sparing the normal organs surrounding the target, in order to minimize radiation side effects . Stereotactic radiation therapy (SRT) is
becoming increasingly available at veterinary facility. SRT delivers an ablative high dose of radiation to a well-defined target with a rapid fall-off of dose, allowing adjacent critical organ
to be spared. It is treated with a limited number of fractions (1–5 treatments) within a same week . The association with current radiation protocols
and molecule markers should be investigated. The limitations of this study include the retrospective nature of the study, a limited case population, and the use of orthovoltage radiation unit. Despite the limitations of this
retrospective study, our results showed that EGFR overexpression could be a prognostic marker in canine nasal carcinoma. Further investigations into the efficacy of RT combined with molecular
inhibitors in veterinary medicine are needed, especially in advanced-stage tumors and those with poor response to RT. 

None of the authors of this paper has any financial or personal relationships that could inappropriately influence or bias the content of the paper. 

