Evaluation of deslorelin implant on subsequent mammary tumors of rats (Rattus norvegicus)

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Evaluation of deslorelin implant on subsequent mammary tumors of rats (Rattus norvegicus) ˜ LV , C. Vergneau-Grosset med vet, IPSAV, CES , L. Pena C. Cluzel med vet, IPSAV, M.Sc. , M.G. Hawkins VMD , E. Maccolini med vet, IPSAV , K. Sinclair DVM , J. Graham DVM , M.J. Sadar DVM , Guzman D. Sanchez-Migallon LV, MS , S. Lair DVM, DES, DVSc , I. Langlois DVM , J. Paul-Murphy DVM PII: DOI: Reference:

S1557-5063(19)30144-2 https://doi.org/10.1053/j.jepm.2019.08.001 JEPM 50247

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Journal of Exotic Pet Medicine

˜ LV , Please cite this article as: C. Vergneau-Grosset med vet, IPSAV, CES , L. Pena C. Cluzel med vet, IPSAV, M.Sc. , M.G. Hawkins VMD , E. Maccolini med vet, IPSAV , K. Sinclair DVM , J. Graham DVM , M.J. Sadar DVM , Guzman D. Sanchez-Migallon LV, MS , S. Lair DVM, DES, DVSc , I. Langlois DVM , J. Paul-Murphy DVM , Evaluation of deslorelin implant on subsequent mammary tumors of rats (Rattus norvegicus), Journal of Exotic Pet Medicine (2019), doi: https://doi.org/10.1053/j.jepm.2019.08.001

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Evaluation of deslorelin implant on subsequent mammary tumors of rats (Rattus norvegicus) Vergneau-Grosset C*, med vet, IPSAV, CES, Peña L, LV, Cluzel C, med vet, IPSAV, M.Sc., Hawkins MG, VMD, Maccolini E, med vet, IPSAV, Sinclair K, DVM, Graham J, DVM, Sadar MJ, DVM, Sanchez-Migallon Guzman D, LV, MS, Lair S, DVM, DES, DVSc, Langlois I, DVM, PaulMurphy J, DVM.

From Service de médecine zoologique (Vergneau-Grosset, Maccolini, Lair, Langlois), the Service de Pathologie (Cluzel), Faculté de médecine vétérinaire, Université de Montréal, Canada, Departamento de Medicina y Cirugía Animal, Anatomía Patológica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain (Peña), Kensington Bird and Animal Hospital (Sinclair), Cummings School of Veterinary Medicine at Tufts University (Graham); Department of Clinical Sciences, Colorado State University College of Veterinary Medicine and Biomedical Sciences (Sadar); and the Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis (Hawkins, Guzman, Paul-Murphy).

*Corresponding author: [email protected]

This study was supported by grants from the Association of Exotic Mammal Veterinarians and from the Center for Companion Animal Health, University of California, Davis. Implants were donated by Virbac Company, Carros, France.

Abstract Background Mammary fibroadenomas are one of the most common tumors of female companion rats (Rattus norvegicus forma domestica). The objectives of this study were to determine if subcutaneous administration of a deslorelin implant following excision of fibroadenomas can prevent or delay development of additional mammary tumors and increase survival in companion rats. Methods Female intact client-owned rats with benign mammary tumors were divided into three groups: no implant (n = 10), placebo implant (n = 10), or 4.7 mg deslorelin implant (n = 10) placed within two months of tumor excision. Rats were monitored for subsequent mammary tumors for ten months following treatment. Expression of estrogen  progesterone, prolactin and androgen receptors stained by immunohistochemistry in primary masses of rats included in the deslorelin-treated group was evaluated using the Allred scoring system. Results In the control non-implanted group, four of the ten rats developed another mammary tumor, including one anaplastic carcinoma. In the control placebo group, five of the ten rats developed another mammary tumor, including one ductal carcinoma. In the deslorelin-treated group, three out of ten rats developed another mammary tumor, including one adenocarcinoma. Median time between surgery and new mass detection did not differ significantly among groups (55 days in the deslorelin group vs. 77 days in the control placebo group, P = 0.25). Median survival times after surgery did not differ significantly among groups. No correlation was noted between receptor expression and response to treatment with deslorelin. Conclusions and clinical relevance Deslorelin implants placed within two months of benign mammary tumor surgical excision were not associated with a decreased risk of developing subsequent mammary tumors, nor with an increased survival in female rats. Further studies are needed to define useful adjunct therapy to surgery. Keywords Fibroadenoma, gonadotropin-releasing hormone agonist, survival, prognosis

Background Mammary tumors are considered to be the most common spontaneous tumors in companion rats (Rattus norvegicus forma domestica) [1]. The majority of these tumors are fibroadenomas [2]. Surgical removal of the mass is currently the treatment of choice, but a second mammary mass often develops within a few months following excision, because complete mammary chain resection is not easily feasible in rats [1] and due to persistent hormonal promoting factors [3]. Of note, neoplastic mass recurrence, sensu stricto is the development of a second mass at the same site or via metastasis of a primary mass [4]. Since rat mammary fibroadenomas are benign, a second mass at a distinct location, strictly speaking, should be called a subsequent mass rather than a recurrence. Chemically induced mammary tumors have been studied more extensively than spontaneous fibroadenomas in laboratory rats: for induced tumors, the recurrence rate after surgical excision varies from 20% [5] to 60% [6] depending on the rat strain and follow-up duration. However, rat induced mammary tumors are mostly carcinomas and adenocarcinomas and have different etiologies [7], thus direct comparison with spontaneous benign mammary tumors is not possible. In a retrospective study of companion rats, more than 75% (12/16) of the rats developed additional mammary masses between 1 and 8 months post operatively with a median of 4.5 months [8]. Surgical sterilization has been shown to reduce the prevalence of spontaneous mammary tumors in laboratory rats when performed prior to tumor development [9,10]. Female rats that underwent ovariectomy at 90 days of age developed significantly fewer mammary tumors than older intact females7 and surgical spay between five and seven months reduced the spontaneous tumor rate from 73.8% to 5.3% [10]. Prophylactic surgical spay is not commonly performed for companion rats [11]. Because companion rats often present to veterinarians for abnormal clinical signs rather than for a wellness examination, there is a need to identify an effective protocol to prevent development of subsequent mammary tumors at the time of surgical excision of the initial mammary tumor. Currently it is unknown whether providing sex hormone-inhibiting drugs after removal of spontaneous mammary tumors from companion rats will decrease or prevent the occurrence of subsequent mammary tumors. The type of mammary tumor and the presence of sex hormone receptors

in mammary tissue will likely impact the effectiveness of various treatments, as reported in women [12]. Circulating concentrations of prolactin and estrogen have been associated with mammary fibroadenoma tumor growth in some laboratory rat strains [13-15]. Prolactin-inhibiting drugs have been shown to prevent spontaneous mammary fibroadenomas [14] and to decrease the size of some laboratory-induced mammary tumors [13, 16-18]; however, recurrence at the same location or new tumor development occurred within one to nine weeks following cessation of treatment [13]. In companion rats, life-long treatments can be challenging. Thus, a contraceptive treatment via a slow release implant is an attractive alternative deserving investigation. Gonadotropin-releasing hormone (GnRH) agonists, such as deslorelin acetate (deslorelin thereafter), act primarily on the anterior pituitary, inducing a transient early rise in gonadotropin release. With slow release formulations, GnRH agonists cause pituitary desensitization or downregulation, leading to suppressed circulating levels of gonadotropins and sex hormones [19]. Gonadotropin-releasing hormone agonists have been shown to induce chemical sterilization in rats [20-26], similar to ferrets, rabbits, dogs, and cats [27]. The deslorelin implant has been shown to down-regulate blood reproductive hormones in female [25] and male rats [24]. No major adverse effects have been associated with deslorelin implants in rats [23]. The latency of action of the 4.7 mg deslorelin implant in female rats is approximately two weeks [23]. The duration of effect of the 4.7 mg deslorelin implant in female rats is greater than a year, as studied by hormonal measurements [25-26], ultrasound and post mortem examination of the reproductive tract [25-26], vaginal smears [23], and by assessment of contraceptive effect [23, 26]. In premenopausal women with hormone receptor-positive breast cancers, treatment with GnRH agonists has been recommended under certain conditions [12, 28]. By extrapolation, the use of deslorelin implants to prevent mammary tumors in companion rats has been previously suggested [27] and performed anecdotally by veterinarians. The first objective of this clinical trial was to determine if subcutaneous (SC) administration of a deslorelin implant in rats following surgical excision of benign mammary tumors can prevent or delay development of additional mammary tumors. The second objective was to evaluate the effect of deslorelin implant on survival after benign mammary mass excision. In the present study, the effect of a GnRH agonist, deslorelin, was evaluated on benign mammary tumors in female rats over the

documented duration of action of the implant, i.e. twelve months. The third objective was to evaluate if expression of estrogen, progesterone, prolactin and androgen receptors was associated with different responses to deslorelin among treated rats.

Methods A multicentric, prospective experimental study was conducted in four veterinary medical teaching hospitals and seven privately-owned veterinary clinics. Rats that presented with a subcutaneous mass were recruited into the clinical trial with the signed owner’s consent. All contributing institutions had IACUC approval and all institutions and practices followed the protocol approved by the University of California, Davis and the Université de Montréal IACUC committees (Protocols 18518 and 15-Rech1785). Subcutaneous masses were measured with a caliper, and diameter in three directions (length, width, depth) was recorded for each mass. Location of the mass was recorded as described in a previous study [8]. After surgical excision and histologic analysis of each mass, only rats with benign mammary tumors were included in the study, and all other rats were excluded.

Surgical procedure: Rats were premedicated with buprenorphine 0.01 - 0.05 mg/kg (Vetergesic, Alstoe, Whitby, ON, USA) or oxymorphone 0.05 - 0.2 mg/kg (Oxymorphone; DSM Pharmaceuticals, Greenville, NC, USA) and midazolam 1- 1.5 mg/kg (Midazolam, Novaplus, Irving, TX, USA or Sandoz, Boucherville, QC, Canada) intramuscularly 30 minutes before induction of anesthesia. Induction was performed with isoflurane (Isoflurane, Piramal Healthcare, Bethlehem, PA, USA or Fresenius Kabi, Lake Zurich, IL, USA) delivered in oxygen in an induction chamber. An antibiotic was administered SC, which varied depending on attending veterinarian preference. Rats were maintained under anesthesia with a tight-fitting facemask and isoflurane concentration was adjusted as needed. Temperature was maintained with warming elements and monitored throughout the procedure. Heart rate was monitored with either one of the following techniques: Doppler probe (Parks Medical Electronics, Aloha, Oregon 97078, USA) placed on a peripheral artery, pulse oximeter, or auscultation. Respiratory rate was monitored during anesthesia using at least one of the following techniques: capnometer, auscultation,

or chest expansion visualization. The area surrounding the mass was shaved and surgically prepared. Lidocaine 2 mg/kg (Lidocaine 2%, Vetone, Boise, ID, USA) was injected SC around the mass for local anesthesia. An elliptical incision was made with a scalpel blade. The mass was bluntly dissected from surrounding tissues and hemostasis was performed with a bipolar electrocautery and vessel ligations with monofilament suture as needed, until the SC mass was excised. The surgical incision was closed with a SC suture, and intradermal suture or tissue glue to avoid exposed sutures. Before anesthetic recovery, the following treatments were administered SC: meloxicam 1 mg/kg (Metacam, 5 mg/ml, Boehringer Ingelheim Vetmedica, Inc. St. Joseph, MO, USA), fluids 30 mL/kg (Lactate Ringer; Baxter). Antibiotics were administered post-operatively according to guidelines [29] and according to clinician discretion. Rats were monitored during recovery in a warm enclosure. In the post-operative period, patients received meloxicam 1 mg/kg (Meloxicam; Boehringer Ingelheim) twice daily orally for five days and an opioid drug as needed for 24-48h. Rats were discharged to their owner and monitored for subsequent mass for a period of twelve months. Any rat lost to follow-up (n = 3), developing a subsequent subcutaneous mass before implantation (n = 1) or treated with cabergoline during the remainder of their lives (n = 2) were excluded from the study and replaced by another recruited rat. Masses were fixed in formalin for at least 48 hours then transferred to ethanol and shipped to the Center for Genomic Pathology Laboratory, University of California, Davis for histopathologic analysis. Fixed tissues were routinely processed and stained with Hematoxylin and Eosin. The slides obtained

were

scanned

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the

high-definition

image

transferred

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(‘spectrum.ucdavis.edu’). Histological characterization was performed by a board-certified pathologist (LP), identifying rats for inclusion in the study, based on previously published histological classification [30]. Mass excision was complete in all cases.

Deslorelin acetate (Suprelorin, Virbac, Carros, France) 4.7 mg implants (n = 10) or placebo implants (n = 10) provided by the manufacturer were randomly assigned to each rat by drawing each implant from a box. Owners were blind to the treatment. Owners who elected to not implant their rats (n = 10) were aware that their rat did not receive an implant.

When applicable, implants were placed SC in the interscapular region. Briefly, each rat was sedated with an opioid such as butorphanol 2 mg/kg (Torbugesic; Fort Dodge Animal Health, Overland Park, KS, USA) SC and midazolam 1 mg/kg SC. An area measuring 1 cm2 was clipped and aseptically prepared, and lidocaine 2 mg/kg was used to block the injection site. The implant was inserted using the sterile needle and the skin was closed with an absorbable simple interrupted cutaneous suture. Rats were monitored during recovery and meloxicam was administered postoperatively at 1 mg/kg PO once a day for five days. Each rat was monitored by owners or the attending veterinarian for subsequent mass for twelve months following mass removal, and date of death was recorded. Adverse effects were recorded, especially any neurologic clinical signs that could be related to the presence of pituitary masses. New masses were excised following the same protocol if elected by the owner. Each new mass was excised either ante or postmortem, and sent to the Center for Genomic Pathology Laboratory, University of California, Davis for histopathologic analysis by the same pathologist (LP). Histologic diagnoses of subsequent masses were recorded. Owners were offered a complete necropsy and results were recorded. Presence of implant was confirmed during necropsies in implanted rats. Primary mammary masses obtained from the ten rats implanted with deslorelin were stained for prolactin (PRL), estrogen a (ERA), progesterone (PR) and androgen (AR) receptors to evaluate if expression of these hormonal receptor was associated with response to deslorelin treatment. Each mass was fixed in 10% neutral buffered formalin for 48 hours, then transferred to 70% ethanol before shipment to the Center for Comparative Medicine of the University of California, Davis. A TissueTek VIP autoprocessor (Sakura, Torrance, CA) was used to process samples for paraffin-embedding. Tissue blocks were then sectioned to 4 µm, sections mounted on glass slides, and samples were processed for immunohistochemistry (IHC) using the following primary antibodies. Rabbit monoclonal antibodies against prolactin receptor (1:500; ab170935, EPR7184(2), Abcam, Cambridge, MA) was used for detection of prolactin receptors as previously described [31]. Rabbit polyclonal antibodies to androgen receptor (1:2000; P21, 06-680, Millipore, Billerica, MA) were used, as previously established by Western blot [32]. Rabbit polyclonal antibodies to ERa (1:1000; sc-542, Santa Cruz Biotechnology, Dallas, TX) were used, as previously established by Western blot [33].

Finally, monoclonal mouse anti-human progesterone receptor (1:500; PgR 636, Dako, Carpinteria, CA) were used as previously established by Western blot [34]. All IHC was performed manually without the use of an automated immunostainer. Antigen retrieval was performed using a Decloaking Chamber (Biocare Medical, Concord, CA) with citrate buffer at pH 6.0, 125°C and pressure to 15 psi. The total time slides were in the chamber was 45 min. Incubation with the primary antibody was performed at room temperature overnight in a humidified chamber. Normal goat serum was used for blocking. Biotinylated goat anti-rabbit (1:1000; Vector Labs, Burlingame, CA) was the secondary antibody used with a Vectastain ABC Kit Elite and a Peroxidase Substrate Kit DAB (both from Vector Labs) used for amplification and visualization of signal, respectively. Tissues known to contain the assessed antigens were used as positive controls and tissues known to not contain the assessed antigen were used as negative controls. Semi-quantitative evaluation of immunohistochemistry staining were performed by a board certified pathologist (CC) using the Allred scoring system as previously described [35, 36]. Scores for nuclei (AR, ER, PR) or cytoplasm (PLR) were calculated according to the percentage of positive cells (0 [0%]; 1 [0.1%-1%]; 2 [1%-10%]; 3 [11%-33%]; 4 [34%-66%]; 5 [67%-100%]) and staining intensity (0 [absent]; 1 [poor]; 2 [moderate]; 3 [intense]). To obtain the Allred score, the proportion and intensity scores were summed to produce total scores. In addition, the Allred score was determined for each mammary tumor epithelial and stromal compartments for each of the four receptors. Results obtained for other mammary masses will be presented in another article.

Statistical analysis Normality was evaluated with a Shapiro-Wilk test. Medians and mean were calculated for nonparametric parameters. Median and mean age for all rats of each group were listed based on rats recorded birth dates and age at the time of surgery, using Excel (Microsoft Excel for Mac, Version 16.16.4, Microsoft corporation, Redmond, WA, USA). Time to subsequent mass emergence, as reported by the owner and attending veterinarian, and time to death were also calculated using date of the first surgery as day zero.

Statistical analysis was performed using R (R Core Team, [2015]. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/, R version 3.1.3). Mass maximal diameter and age were compared among all three groups using a Kruskal-Wallis test. Rates of subsequent mammary tumor development were compared among groups with a two-tailed Fisher’s exact test. Time to new tumor development (disease-free survival, DFS) was compared two by two among groups using a Wilcoxon rank sum test. Total Allred scores were compared between rats of the deslorelin-treated group who developed a subsequent mass versus rats of the deslorelin-treated group who did not develop a subsequent mass for each evaluated hormonal receptor. Time to death (overall survival) was compared using a Kaplan Meier test among all groups and between the groups receiving deslorelin and the control placebo group. The significance level was set at 0.05.

Results A summary of initial presentation is presented in Table 1. Age of rats in the deslorelin-implanted group, in the control placebo group and in the control non-implanted group were not significantly different (median: 18 vs. 18 vs. 22 months of age; mean: 17 vs. 18 vs. 20 months of age, P = 0.29;). Initial masses were: seven fibroadenomas, two lactating adenomas, and one tubular adenoma with multifocal fibroadenomas in the deslorelin-treated group; seven fibroadenomas, and three mammary fibromas in the control placebo group; and seven fibroadenomas, two lactating adenomas, and one mammary fibroma in the control non-implanted group (Figure 1). Mass median maximal diameter was respectively 3.5 cm in the deslorelin-treated group, 5 cm in the control placebo group, and 3.5 cm in the control non-implanted group. Mass mean maximal diameter was respectively 3.3 cm in the deslorelin-treated group, 6.0 cm in the control placebo group, and 3.9 cm in the control non-implanted group. Mass maximal diameter was not significantly different among groups (P = 0.31). Time between surgical excision of the mass and implant placement ranged between 21 and 50 days. Overall, twelve of thrity rats developed subsequent mammary tumors while eighteen of thrity rats were disease free. In eight of the twelve recurring mammary masses, the location was different from the primary mass (see Table 1). One of the twelve rats who developed subsequent mammary masses

had a second mass excision. Three of the twelve rats who developed subsequent mammary masses were euthanized due after mass detection. Deslorelin implants placed within two months of benign mammary tumor surgical excision did not prevent subsequent mammary tumors (4/10 vs. 5/10 vs. 3/10, P = 0.69) when compared to the two control groups. In the control non-implanted group, four of ten rats developed subsequent mammary tumors including one anaplastic carcinoma and a low-grade carcinoma. In the control placebo group, five of ten rats developed subsequent mammary tumors, including one ductal carcinoma. In the deslorelin-treated group, three of ten rats developed subsequent mammary tumors, including one adenocarcinoma (see Table 1). Time to new tumor development (DFS) was not significantly different among the groups (median: 55 vs. 77 days; mean: 77 vs. 121 days, P = 0.25). When comparing groups two by two, DFS was not significantly different between deslorelin-treated and control non-implanted groups (median: 55 vs. 155 days; mean: 77 vs. 152 days, P = 0.23), nor between deslorelin-treated and control placebo groups (median: 55 vs. 77 days; mean: 77 vs. 96 days P = 0.25). Allred scores for ERA PR, PRL and AR receptors in primary masses of rats from the deslorelintreated group are presented in Table 2. When comparing rats that developed subsequent masses (n = 3) or not (n = 7) in the deslorelin-treated group, no difference was noted in expression of PR, AR, ERA nor PRL receptors (P values are included in Table 2). A summary of the cause of death or euthanasia and necropsy results obtained in three cases can be found in Table 1. Overall survival was not significantly different between the deslorelin group and the control placebo group (222 vs. 137 days, P = 0.17) nor among the three groups (P = 0.33). Median survival time after surgery was 222 days in the deslorelin-treated group versus 148 days in the control groups (137 days in the control placebo group, and 185 days in the control non-implanted group). Mean survival time after surgery was 254 days in the deslorelin-treated group versus 180 days in the control groups (147 days in the control placebo group, and 213 days in the control non-implanted group).

Conclusions and clinical relevance

In this study, the use of deslorelin implants was not associated with a decrease in the risk of developing subsequent mammary tumors after surgical excision of spontaneous benign mammary tumors in this small population of 30 companion rats. In contrast, in another study, treatment with a GnRH analog for twenty days led to regression of carcinogen-induced mammary tumors in laboratory rats [37]. However, these mammary masses were adenocarcinoma, contrary to the tumors of the present study. Leuprolide, another GnRH agonist, has also been shown to prevent carcinogen-induced mammary tumors in rats when injected daily starting two weeks prior until one week after carcinogen administration [38]. Dimethylbenzanthracene-induced and N-Methyl-N-nitrosourea-induced mammary tumors are well studied endocrine-dependent malignant mammary tumors of rats. Rat mammary carcinomas induced with these carcinogens are strongly estrogen and progesterone receptors positive [39] and show strong and relatively consistent immunohistochemistry staining for androgen and prolactin receptors, while spontaneous fibroadenomas had weak to moderate immunoreactivity [40]. Conversely, the prevalence of hormonal receptors in spontaneous benign mammary tumors of companion rats has not been evaluated to date. Given the results of this study, it is possible that companion rat spontaneous benign mammary tumors are not strongly endocrinedependent, as has been reported in certain laboratory rat strains [40]. In canine experimental studies, GnRH agonists inhibited the growth of hormone-dependent mammary tumors and have been suggested as an adjunctive treatment of mammary carcinomas [41, 42]. Conversely, GnRH agonists are not indicated as a single adjunctive therapy of mammary, however may be recommended in conjunction with tamoxifen, a selective estrogen receptor modulator, for hormone-receptor positive cancers in premenopausal women [12]. Similarly, it might be relevant to study hormonal receptors in rats and to evaluate whether combined treatment with tamoxifen and a GnRH agonist would decrease or delay subsequent mammary tumors. An initial postulation regarding the cause of subsequent mammary tumor occurrence in rats from the deslorelin-treated group, was the prolonged time between surgical removal of the initial mammary tumor and placement of the deslorelin implant. The study design was to implant only rats bearing benign mammary tumors, therefore implantation was delayed to obtain histologic diagnosis. For this reason, four additional female rats with mammary fibroadenomas were implanted with

deslorelin at the time of mass excision at the owners’ requests. Two of these four rats developed subsequent mammary masses, a lactating adenoma in one case, and a tubular carcinoma in the other case. Although more cases would be needed to conclude with certainty, deslorelin implant did not prevent subsequent mammary masses when placed at the time of surgical excision in these four cases. More studies are needed, including more cases and possibly selecting mammary tumors displaying certain receptors; implants should be placed at the time of surgery in further studies to evaluate if this protocol decreases the occurrence of subsequent mammary tumors. Prophylactic effect of early ovariectomy [9] and anti-prolactin treatments [13] on spontaneous mammary tumor development has been well documented in rats. However, these effects were likely mediated through the inhibition of mammary tissue development [13], as late ovariectomy was shown to decrease mammary tumor prevalence to a lesser extent than early ovariectomy [10]. It is unknown whether ovariohysterectomy (OVH) at the time of mammary tumor excision decreases the prevalence of subsequent mammary tumors. Since deslorelin implants placed at the time of benign mammary mass excision did not prevent subsequent mammary tumors, it is suspected that surgical OVH or ovariectomy might not have a prophylactic effect either. In dogs, OVH at the time of benign mammary mass surgical excision does not generally prevent new mammary mass development, but certain dogs with mammary tumors expressing estrogen receptors may benefit from OVH [43]. Regarding subsequent mammary masses, various histologic diagnoses were obtained and only eight out of twelve (67%) subsequent masses were fibroadenomas. As previously mentioned, this supports that additional mammary masses should not be termed “recurrences.” Malignant mammary tumors were observed in all three treatment groups. There is debate regarding whether fibroadenomas and adenocarcinomas are a continuum of the same disease process in rats [44], although mammary adenocarcinomas can arise concomitantly with mammary fibroadenomas or following their excision. In this study, it was elected to only enroll rats with benign mammary tumors to have homogenous treatment groups. However, it should be acknowledged that only a section of the excised mass was examined histologically, as would be done routinely for clinical cases. Therefore, rats with focal areas of malignant tumors within their mammary fibroadenomas may have gone undetected if this tissue was not included in the histologic section. However, this confounding factor would be present for all

three groups. Complete excision of the primary mass was noted in all cases and subsequent masses were recorded in different anatomic location in eight out of twelve cases. This confirms that these subsequent masses did not arise from incompletely excised tissue, and rather developed from another mammary gland. The presence of non-detected mammary tumors at the time of surgical mass excision cannot be ruled out. This confounding factor would also be present in all three groups, and the study remains representative of a clinical situation where some mammary tumors in development would not be detected at the time of surgery. No differences in ERA PR, PRL and AR receptors expression was noted between primary masses of rats that did, or did not have subsequent masses in the deslorelin-treated group. However, a higher number of rats would be needed to rule out a correlation between expression of these receptors and response to deslorelin. It should be acknowledged that receptor expression does not necessarily mean that these receptors are functional [45]. The present study is the first one to evaluate hormonal receptor expression in companion rat mammary tumors and results would need to be confirmed in future studies. Further studies should also include molecular techniques to evaluate transcription of hormonal receptors in spontaneous rat mammary tumors. In addition, it would be useful to compare expression of hormonal receptors in rat mammary tumors versus normal mammary tissue to evaluated which receptors are upregulated in neoplastic tissue. Rats implanted with deslorelin did not have a shorter life span than rats from the control groups. In addition, development of neurologic signs was similar in the deslorelin-treated group as in the control groups. Since deslorelin has an initial stimulatory effect on the pituitary gland and may cause death in humans with pituitary tumors [46-51], it was important to evaluate this potential adverse effect in this geriatric population of companion rats, as pituitary gland tumors are common in geriatric rats [8]. In some laboratory rat strains, pituitary adenomas have been described in more than 80% of rats older than 24 months of age, including the Sprague-Dawley [52], Fisher [53] and Wistar rats [2,54]. Pituitary tumors have also been documented in 75% of companion rats with mammary fibroadenomas in a retrospective study [8]. Of note, no causation between pituitary prolactinoma and mammary tumor development has been established in rats [9]. In these cases, prolactin-secreting pituitary tumors are found concomitantly with mammary fibroadenomas, but it is undetermined

whether it is coincidental given the prevalence of both tumors in geriatric rats [1,9]. Regardless, this study demonstrated that deslorelin implants are not contraindicated in geriatric rat patients, although no preventive effect was found on mammary tumors. Survival time was not significantly longer in the group receiving a deslorelin implant than in the group receiving a placebo implant. Conversely, treatment with a GnRH agonist, goserelin, in female dogs with mammary carcinomas, for twelve months was associated with an increased survival time compared to controls in a study [41]. This difference may be due to different hormonal receptors in rat mammary fibroadenomas compared to dog mammary carcinomas. Further studies would be needed to investigate hormonal receptors of rat mammary tumors.

Limitations of this study include the small number of rats, which could have caused a type II error. Both median and mean were included in the result section as median are considered informative for small sample sizes [55]. Further studies should include more cases. Subsequent mammary masses were detected by owners, which could have influenced the time between surgery and new mass detection recorded in the present study. However, this bias was present in all groups. Recorded survival times were influenced by owner decisions as certain rats were euthanized. In addition, a nonblinded group of ten non-implanted rats was recruited for the study to increase statistical power in a context where rat owners may not be willing to try a new drug on their companion animal. Further studies are needed to investigate the effects of prolactin antagonists and multimodal therapy such as combining GnRH agonists with tamoxifen, as advocated in premenopausal women diagnosed with breast cancer.

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 the paper.

Acknowledgements This project was supported by a Research grant from the Association of Exotic Mammals Veterinarians and by the Center for Companion Animal Health, University of California, Davis. Placebo and deslorelin implants were kindly provided by Virbac Company, Carros, France. The authors would like to thank Guy Beauchamp (Université de Montréal) for statistical advice, Dr. Amy Wells (Avian and Exotic Clinic of Monterey), Steven Liston (Liston Animal Hospital), Erica Giles (Kensington Bird and Animal Hospital, CT, USA), Winifred Krogman (Northside Animal Hospital, NH, USA), Noémie Summa (Université de Montréal), Sarah Gardhouse, Molly Gleeson (University of California, Davis), Dan Famini (Petcare Veterinary Clinic, California, USA), Lucile Chassang, Thomas Coutant (Veterinary Hospital Frégis) for taking care of rats included in this project, and Neil Hubbard (Center for Genomic Pathology Laboratory) for mass staining and scanning.

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Figure 1: Histology of companion rat primary mammary tumors (Hematoxylin and Eosin stain; bar = 50 m): A: tubular adenoma (magnification 20x), B: lactating adenoma. (magnification 20x), C: fibroma (magnification 20x), D: fibroadenoma (magnification 20x).

Table 1: Summary of presentation at the time of inclusion, outcome, and cause of death of 30 rats included in the experimental trial Outcome

Inclusion

Case

Age at Histologic inclusion diagnosis (months)

Location of the primary mammary tumor

MMD (cm)

Time between surgery and deslorelin implant placement

Time between surgical Time between Histologic excision and mass detection diagnosis of subsequent mass and second subsequent detection surgery masses (days)

Location of subsequent mammary masses

Survival time after surgery (days)

Clinical presentation before death

Necropsy results

Deslorelin-implanted group

1

18

Fibroadenoma

2

18

Fibroadenoma

3

18

Fibroadenoma

4

12

5

Right axilla and left inguinal area Right axilla

4

28

NA

NA

417

Found dead

NA

1.2

28

NA

NA

394

Dyspnea

NA

Left axilla

2.8

28

55

115

Fibroadenomas Right axilla and and multiple cystic 296 right inguinal area adenomas

Dyspnea

Chronic progressive nephropathy, bronchopneumonia, pituitary adenoma

Fibroadenoma

Right axilla

2.9

21

28

NA

Dyspnea

Hemic pulmonary neoplasm and histiocytic bronchopneumonia

18

Tubular Adenoma with multifocal fibroadenomas

Right axilla

5.2

29

NA

NA

235

Neurologic signs

NA

6

21

Fibroadenoma

Right axilla

5

40

NA

NA

208

Dyspnea

NA

7

12

Adenoma

1

50

NA

NA

198

Dyspnea

NA

NA

Adenocarcinoma Left inguinal area 184 and fibroadenoma

Neurologic signs

NA

8

12

Fibroadenoma

Left axilla Right inguinal area

2

41

147

Fibroadenoma

Left inguinal area 293

9

18

Lactating Adenoma

Left axilla

3

39

NA

NA

174

Found dead

NA

10

24

Fibroadenoma

Left inguinal area

5

50

NA

NA

142

Found dead

NA

1.5

NA

198

NA

434

Found dead

NA

5

NA

NA

NA

Dyspnea

NA

6.5

NA

NA

NA

Found dead

NA

2.6

NA

116

NA

Fibroadenoma

Left axilla

152

Dyspnea

Abscessing bronchiectasis and otitis media

10

NA

34

NA

Fibroadenoma

Right inguinal area

144

Neurologic signs

NA

5.7

NA

NA

NA

130

Neurologic signs

NA

3

NA

69

NA

Fibroadenoma

Right axilla

Dyspnea

NA

2

NA

62

NA

Ductal carcinoma

Right inguinal area

Euthanized due to subsequent mass

NA

2.8

NA

NA

NA

Found dead

NA

10

NA

NA

NA

4

Found dead

NA

1.5

NA

NA

NA

645

Dyspnea

NA

Control placebo group 1

12

Fibroma

2

18

Fibroadenoma

3

12

Fibroadenoma

4

24

Fibroadenoma

5

26

Fibroadenoma

6

19

Fibroadenoma

7

18

Fibroma

8

18

Fibroadenoma

9

23

Fibroadenoma

10

12

Fibroma

Left inguinal area Right axilla Right inguinal area Right axilla Right inguinal area Right inguinal area Right axilla Right axilla Bilateral cervical area and left axilla Left inguinal area

Fibroadenoma

Right axilla

264 152

82 62 42

Control non-implanted group 1

22

Fibroadenoma

Right axilla

Left ventrum

2

24

Fibroma

3

18

Lactating Adenoma

4

27

Fibroadenoma

5

22

Fibroadenoma

Right axilla Right ventrum Left axilla

6

24

Lactating Adenoma

Right axilla

7

24

Fibroadenoma

8

18

Fibroadenoma

9

12

Fibroadenoma

10

12

Fibroadenoma

NA: Non Applicable MMD: Mass Maximal Diameter

Left ventrum Right inguinal area Right axilla Right axilla

Fibroadenomas and low-grade carcinoma

Euthanized due to subsequent mass and dental malocclusion

NA

271

Found dead

NA

233

Neurologic signs

NA

201

Hemoptysis

NA

Right axilla

169

Found dead

NA

Left axilla

77

Euthanized due to subsequent mass

NA

NA

62

Dyspnea

NA

NA

NA

60

Neurologic signs

NA

NA

NA

31

Neurologic signs

NA

1

NA

257

NA

2.5

NA

NA

NA

2

NA

233

NA

4

NA

NA

NA

4

NA

41

NA

Lactating adenoma

8

NA

77

NA

Anaplastic carcinoma

8

NA

NA

1

NA

3

NA

Fibroadenoma

Bilateral axillary 377 masses

Left ventrum

Table 2: Allred score of primary masses obtained from rats treated with deslorelin, evaluated for epithelial and stromal compartments for progesterone, androgen, estrogen  and prolactin receptor expression stained by immunohistochemistry. Allred scores were compared using a Wilcoxon rank sum test between rats who did or did not develop a subsequent mammary mass.

PROGESTERONE RECEPTOR Epithelial cells Stromal cells Cell Cell Sta Al Sta Al per per ini lr ini lr cen cen ng e ng e tag tag ID Int d Int d e e en sc en sc stai stai sit or sit or nin nin y e y e g g Rats who developed a subsequent mass 3 2 3 5 0 0 0 4 2 3 5 0 0 0 8 1 3 4 0 0 0 Rats who did not develop a subsequent mass 1 2 2 4 0 0 2 3 2 5 0 0 5 2 2 4 0 0 6 2 2 4 0 0 7 2 3 5 0 0

0 0 0 0 0

ANDROGEN RECEPTOR Epithelial cells Stromal cells Cell Cell Sta Al Sta Al per per ini lr ini lr cen cen ng e ng e tag tag Int d Int d e e en sc en sc stai stai sit or sit or nin nin y e y e g g

ESTROGEN RECEPTOR  Epithelial cells Stromal cells Cell Cell Sta Al Sta Al per per ini lr ini lr cen cen ng e ng e tag tag Int d Int d e e en sc en sc stai stai sit or sit or nin nin y e y e g g

PROLACTIN RECEPTOR Epithelial cells Stromal cells Cell Cell Sta Al Sta Al per per ini lr ini lr cen cen ng e ng e tag tag Int d Int d e e en sc en sc stai stai sit or sit or nin nin y e y e g g

0 0 0

0 0 0

0 0 0

0 3 0

0 1 0

0 4 0

2 4 4

1 2 1

3 6 5

3 4 4

2 2 1

5 6 5

5 5 5

2 2 1

7 7 6

0 0 0

0 0 0

0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 3 0 2 0

0 1 0 1 0

0 4 0 3 0

4 2 4 4 5

1 2 1 2 2

5 4 5 6 7

3 2 5 4 1

1 2 3 2 1

4 4 8 6 2

4 5 5 4 5

1 2 1 1 2

5 7 6 5 7

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

9 10

P val ue co mp arin g the two sub set s

2 3

3 3

5 6

1

0 0

0 0

0 0

N A

0 0

0 0

0 0

N A

0 0

0 0

0 0

0. 8 9

5 3

3 1

8 4

0. 5 6

4 1

3 1

7 2

0. 7 3

5 5

1 1

6 6

0. 2 7

0 0

0 0

0 0

N A