Clinical application of the MEK inhibitor trametinib in dogs with oral squamous cell carcinoma

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Katt, Cheryl E. Balkman, Michael Byron, Patrick C. Carney, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6821917/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: Oral squamous cell carcinoma (OSCC) is a common and deadly disease in dogs. The current standard of care involves wide-margin surgical excision of tumor tissue, which is frequently disfiguring and can be debilitating, leading to a reduced quality of life. Recent studies have demonstrated that OSCC in dogs typically shows highly elevated RAS signaling compared to healthy gingival tissue. Here, we demonstrate that the FDA-approved drug trametinib, which is prescribed in humans for BRAF -mutant melanomas, is effective in treating OSCC in dogs. Methods: Domestic companion dogs (N = 20) with spontaneously occurring OSCC tumors were recruited over a two-year period for an interventional study without concurrent controls. Dogs were prescribed 0.015 to 0.035 mg/kg trametinib daily to be given orally, and owners monitored their dog’s health at home. Treatment was continued for 8 weeks, with examinations every 2 weeks. Tumor volume was assessed by caliper measurement and by computed tomography (CT) imaging. Results: Five dogs achieved a partial response (PR), and one dog had a complete response (CR) based on CT imaging at the end of 8 weeks of treatment, however cancer cells were detected by histological examination. Four dogs had stable disease (SD), and ten dogs were removed from the trial after demonstrating progressive disease (PD). Response categorization was based on R.E.C.I.S.T. criteria, and PD was typically detected by caliper measurements within 2-4 weeks of beginning treatment. The overall response rate was 50% (30% CR/PR, 20% SD). Adverse events were rare, low grade, and resolved with outpatient supportive care. Conclusions: Trametinib effectively blocks the growth of canine OSCC in ~50% of dogs and is well tolerated with minimal side effects. Approximately one-third of the dogs treated showed at least partial tumor volume decrease. Considering all of our findings together, we determine that trametinib is a plausible neoadjuvant step for OSCC which shows clinical benefit for approximately half of dogs prior to surgical intervention. Overall, this work presents an effective, safe, and available targeted therapeutic approach for the treatment of OSCC tumors in dogs. Cancer oral squamous cell carcinoma canine dog trametinib RAS signaling clinical trial Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Oral tumors in dogs are relatively common, affecting an estimated 0.5% of dogs in their lifetimes 1 , and representing 6% of the total cancer burden in dogs 2 . One of the most common oral canine tumors is oral squamous cell carcinoma (OSCC), an aggressive and rapidly growing disease of epithelial origin that makes up ~ 19% of all oral tumors in dogs 3 . Ki67 staining of tumor sections shows relatively high proliferative rate compared to other common oral tumors such as canine acanthomatous ameloblastoma (CAA) 4 . OSCC is also metastatic, with up to 20% of dogs showing signs of metastasis at the time of diagnosis 2 , 5 , 6 . OSCC in dogs is locally invasive and grows in multiple anatomical planes, which can cause severe pain and dysfunction, and in some cases leads to more severe morbidity including pathological fracture 7 . In the last decade, canine OSCC has been the focus of significant research which has helped to define the molecular landscape of the disease. Crucially, recent findings have shown that OSCC has highly activated RAS signaling (Fig. 1 A) 8 – 12 . RAS is a small GTPase which is activated by extracellular signaling through transmembrane receptors, and its activation initiates multiple signaling cascades supporting cell growth and survival 13 – 18 . However, the most important of these in the context of the sustained growth necessary to support a tumor is the RAS-RAF-MEK-ERK pathway (Fig. 1 B). Despite recent advances in understanding the molecular mechanisms driving OSCC, the approach to treatment in canine patients has not yet seen any benefit. The standard of care is largely centered around wide-margin surgical excision of the tumor (Figs. 1 C, 1 D), with radiotherapy being occasionally employed to attempt to shrink or halt the growth of a tumor which is not surgically treatable, or as an adjuvant treatment to surgery 2 , 19 , 20 . While this surgical approach is highly effective, with very low recurrence rates when tumors are completely excised before becoming metastatic, the treatment is also highly invasive, invariably resulting in facial disfiguration and leading to difficulty eating in ~ 30% of dogs 21 . Moreover, larger surgeries lead to both longer healing times and greater chances for postoperative complications and pain 2 , 19 , 22 – 24 . These realities led us to investigate whether the RAS pathway could be targeted to shrink OSCC tumors. While gene set enrichment analysis clearly indicated the upregulation of RAS signaling in OSCC 25 , it is currently unclear which mutations lead to this activation. RAS itself does not appear to be mutated in most OSCC, despite being frequently mutated in CAA 8 . The RAS signaling partner BRAF is mutated (V595E, corresponding to the constitutively activating V600E mutation in humans), but only in about a third of OSCCs, predominantly those of the papillary subtype 10 , 26 . Because it is unclear if a single mutated protein is common to all, or even most, OSCC in dogs, when we set out to find a targeted therapeutic approach to OSCC we prioritized drugs which worked on wildtype proteins and did not require a mutation to bind. This ruled out the promising new RAS inhibitors such as sotorasib, which only tightly bind to mutant RAS proteins, but left several drugs available, including the MEK inhibitor trametinib 17 , 27 – 29 . Trametinib (Fig. 1 E) is a small molecule which inhibits MEK1 and MEK2 30,31 . It binds to the non-phosphorylated version of either protein, blocking their catalytic activity and thus their ability to activate ERK. The drug occupies an allosteric pocket close to the ATP binding pocket on MEK, and crystal structures show that it not only directly blocks binding of BRAF to MEK, thus preventing MEK phosphorylation, but also seems to stabilize binding of MEK to KSR, the kinase suppressor of RAS, which presumably acts to further reduce the binding affinity of MEK for BRAF 32 . Trametinib has been extensively studied, with a well verified safety profile 33 , and it is approved in the US by the FDA for the treatment of BRAF-mutant melanoma or non-small cell lung cancers in humans, in which case it is used alongside the BRAF inhibitor dabrafenib 30 , 34 . Further, trametinib has high oral bioavailability in both dogs 33 and humans 30 , 35 , so can be taken at home. We have shown that it is effective at reducing the growth of canine OSCC cell culture models and murine xenograft models 25 . The objective of this study was to test the hypothesis that daily treatment with trametinib can shrink OSCC tumors in domestic dogs while being well tolerated at effective doses. Dogs with spontaneously occurring OSCC were treated for 2–8 weeks with trametinib. Tumor volumes were determined by contrast-enhanced computed tomographic (CT) scanning before and periodically during treatment. We found that 50% of dogs experienced a decrease in tumor volume, or had the tumor stop growing, over the course of treatment. Methods The study design was reviewed and approved by Cornell University’s Institutional Animal Care and Use Committee, protocol number 2023-0034. Study design and approval was in place before animal recruitment began. Dogs were eligible for enrollment if they had an OSCC, as confirmed histologically by the Animal Health Diagnostic Center (AHDC) at Cornell University, arising from a mucosal surface, were able to undergo at least two rounds of general anesthesia, and could visit the clinic at least every two to four weeks during the study period. Dogs were ineligible if they were pregnant or lactating, their oral tumors had been previously excised, they had previously received radiation or chemotherapy, they were less than one year old, or they had signs of metastatic disease or of any unrelated chronic debilitating illness. Exclusion diseases included diabetes mellitus, uncontrolled Cushing’s or Addison’s disease, renal insufficiency, congestive heart failure, and any other cancer. Following enrollment (day 0), oral trametinib was immediately prescribed (0.015 mg/kg to 0.035 mg/kg once daily, with dose escalating between dogs when three prior patients had been given a lower dose and found to tolerate it with no adverse effects, as detailed below). Trametinib was sourced from the Best Pet RX Pharmacy as a formulation including dimethyl sulfoxide and was given to the dog by the owners at home. Owners were given information concerning drug handling and use, and a list of common side effects along with a questionnaire to document any adverse events from drug treatment. Drug response and tumor progress were monitored during biweekly physical examinations of each dog. These examinations included tumor measurement with calipers, a complete blood count, and a serum biochemistry panel. Caliper measurements were conducted in three planes whenever possible, and in two planes when a third plane could not be reasonably identified. Full tumor staging was conducted on days 0, ~ 28, and ~ 56. Staging included a cytological and/or histological assessment of regional lymph nodes to check for metastasis, and a contrast-enhanced (2mL/kg iohexol, 350 mg/mL iodine) CT head exam, using a 16-slice Aquilion LB helical CT (Canon Medical Systems USA, Inc., Tustin, CA) to assess the main tumor burden. The head CT examination included general anesthesia using standard-of-care individualized protocols determined by a board-certified veterinary anesthesiologist, and was conducted by acquiring continuous axial slices, with a slice thickness of 0.5 mm to 2.0 mm and volumetric reconstructions with effective slice thicknesses of 0.3 mm to 1.0 mm. The CT imaging was analyzed by a board-certified veterinary radiologist as DICOM studies in the hospital’s Picture Archiving and Communication System (Carestream VuePACS, Rochester, NY, USA) using multiplanar reconstructions in bone and soft tissue windows, pre- and post-contrast. The tumor volume was determined by measuring the cross-sectional area of the abnormal/tumor tissue in each slice and multiplying by the slice thickness. Each 2-week examination was used as a decision point for continuing trametinib treatment. If progressive disease was noted, treatment was discontinued, and the dog was shifted to the normal standard of care, as determined by the attending veterinarian. Guidelines for managing side effects were developed; mild adverse events were to be managed symptomatically, while moderate or severe problems would result in decreasing the drug dose or discontinuing the study at the discretion of the attending veterinarian. The experimental unit in all reported data is a single dog being treated, and only domestic companion animals with naturally occurring OSCC were used in this study. No experimental animals were used to supplement enrollment numbers. All dogs received treatment, no negative control group was included due to anticipated enrollment numbers and general understanding of the growth rate of untreated tumors. The total experimental cohort was twenty dogs. Because no negative control was included, it was impossible to blind study participants as to the nature of treatment. However, all data was analyzed by an investigator unaware of which dog’s data they were working on, then compiled by another investigator who could match the data to the dog. No efforts were made to ensure gender balance or a balance between tumor origin sites. Dose escalation The starting dosage was 0.015 mg/kg, and dosages were escalated in increments of 0.005 mg/kg in cohorts of at least three dogs. Toxicity was monitored with complete blood counts and chemistry panels every 2 weeks. Gastrointestinal toxicity was assessed from medical histories reported by owners. If none of the dogs treated in a given cohort experienced a severe toxicity (Grade 3 or higher adverse event), the dosage for the next cohort was escalated. Cohort size varied in order to accumulate necessary safety data before escalating the dose in a new cohort. If one dog experienced severe toxicity, at least three additional dogs were treated at that dosage. If no additional dogs experienced severe toxicity at that dosage, then the escalation continued. If ≥ 2 dogs in a cohort experienced severe toxicity, additional enrollment in that cohort was stopped. Data analysis and statistical approach The cohort size of 20 dogs was supported by a power calculation performed as a paired t-test using PROC POWER in SAS 9.4. The power calculation assumed tumors averaging 20–40 mm in diameter, a correlation of 0.5, and an alpha of 0.5, and suggested that 14–15 dogs at a minimum would be required for an adequately powered study, based on tumor size before and after treatment. CT measurements were considered to be more accurate than caliper measurements and so were used when possible as the basis for determining R.E.C.I.S.T. categorization of treatment, and for all graphs showing the difference between tumor volume at start and exit of the study. All CT data reported show the exact volume determined for each dog. Because these volumes were available for dogs that completed the study, we used them rather than ‘longest axis’ measurements to determine R.E.C.I.S.T. categorizations. Specifically, CR was a tumor that could not be detected by CT. PR was a tumor which had a volume at least 30% smaller than the original volume. SD was a tumor with less than 30% reduction or 20% increase in volume compared to the original tumor volume. And PD was a tumor which grew more than 30%. For dog #10, no exit CT scan was conducted, so PD was based on estimated volume from caliper measurements. Univariate tests based on the degree of tumor volume change over the course of treatment were conducted to analyze differences in treatment success rate between various groups. Statistically significant differences between compared groups were determined using tests appropriate for independent samples that are not normally distributed, namely the Mann-Whitney U test (for two groupings) or Kruskal-Wallis test (for three or more groupings). These calculations were completed in GraphPad Prism 10.4.2. Differences in results between groups were considered to be statistically significant at a level of p ≤ 0.05. Caliper measurements were used to follow tumor size over the course of the experiment and are used for graphs showing tumor growth over time. Where CT scans are shown in the data, the shown slice is at or near the thickest part of the tumor as determined by the veterinary radiologist. All twenty dogs are included in every analysis reported. Results Twenty dogs were enrolled over a period spanning from October 2023 to February 2025 after meeting the inclusion criteria. Dogs either initially presented to the Cornell University Hospital for Animals (CUHA) or were referred by their primary practitioners . All dogs were ultimately enrolled in the veterinary clinical trial following a visit to CUHA, and all reported examinations were performed at CUHA except as indicated. Dogs were diagnosed with OSCC following routine tumor staging and histological workup 4,8,36 ( Figure 2 ). The dogs included fifteen purebreds, with the remaining being various mixed breeds ( Table 1 ). The cohort included nine spayed females (45%) and eleven castrated males (55%). Ages ranged from 3 to 14 years with a median of 9 years. Eight tumors were located on the caudal maxilla, five on the rostral maxilla, four on the rostral mandible, two on the caudal mandible, and one was sublingual. Initial tumor volume, as determined by CT, ranged from 0.27 cm 3 to 71.62 cm 3 , with an average initial volume of 23.7 cm 3 . Histological examination showed 3 tumors to be basaloid, 6 to be papillary, and 11 to be conventional, further broken down as 5 with poor differentiation, 5 with moderate differentiation, and 1 well differentiated 10,37 . All twenty histological subtypes were determined by the same pathologist. After histologic confirmation of OSCC and staging, dogs were prescribed trametinib, to be given orally by the owners once daily. Tumors’ response to drug treatment was classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD, consistent with widely-used R.E.C.I.S.T. reporting parameters 38 . As shown in Table 2 , 6 dogs exhibited CR or PR (overall response rate [ORR] 30%), while fourteen dogs exhibited SD (20%) or PD (30%) with treatment. Figure 3A and Table S1 show the tumor volume before and after treatment for each patient, while Figure 3B shows the percentage growth or reduction in size for each patient’s tumor, calculated as (exit volume – initial volume)/(initial volume)*100. One dog (#11) was removed from the trial due to obvious tumor growth before a 1-month CT scan could be conducted, and the tumor was estimated to have roughly doubled in size by visual assessment. For all other dogs, final tumor volume was determined by CT when they exited the study. While our trial was designed for 8 weeks of treatment, some tumors responded to the drug within 2 weeks. Indeed, for five dogs their tumor volume had been reduced by the date of their first post-treatment evaluation (average of 45% tumor volume reduction, average of 18 days between first and second examination). For dog #8, the tumor began to regrow by the next examination. The tumors of several dogs had almost entirely disappeared after 2-6 weeks of treatment ( Figure 4A ), which in some cases was easily apparent by external visual inspection ( Figures 4B-4E ). In patients where CR or PR was observed, we examined their CT scans to determine any other anatomical results from treatment. The soft tissue scans easily demonstrated a reduction in tumor volume ( Figures 5A, 5C ), and bone window scans showed rapid bone regrowth in some (N=2) responsive patients ( Figures 5B, 5D ). Physiological changes due to treatment Patient health was monitored at home by the owners and was reported via a survey to document adverse events ( Supplemental information, Document 1 ). No high-grade adverse events were detected in any of the dogs or reported by clients, although ~30% of owners did report that their dogs had transient flatulence that resolved spontaneously after a few days. Also, per one dog’s owner, that dog exhibited two vomiting episodes a few days after initiating therapy, although the animal had a history of chronic vomiting so these events may or not have been related to trametinib administration. None of the dogs had to be removed from the study and none of the dosing regimens had to be modified due to observed or reported adverse events. Patients were also examined every ~two weeks at CUHA or by the primary practitioner in consultation with CUHA clinicians. Routine bloodwork showed no significant adverse events from the drug. Table 2 summarizes several key values, namely neutrophil count (NEU), liver enzyme expression (alanine transaminase/ALT, aspartate transaminase/AST, and alkaline phosphatase/ALP), and markers of renal function (blood urea nitrogen/BUN, creatinine). Three dogs had their pre-treatment bloodwork completed at facilities which do not routinely measure AST levels, and these are marked as not measured (n.m.). While several dogs had assorted values that fell outside the normal range, post-treatment measurements were typically no worse than pre-treatment measurements, and represented predominantly grade 1 and grade 2 adverse events, which are summarized in Table 3 39 . Several grade 1 events were only marginally outside the normal expected range. Only two grade 3 metabolic abnormalities occurred (ALP, dogs #4 and #20) and each dog had grade 2 pre-treatment elevations in ALT and/or ALP, suggesting preexisting liver pathology unrelated to trametinib treatment. BUN and creatinine levels were only marginally outside the normal range for three dogs (#14, #17, and #18). No dog showed clear signs of neutropenia before or after treatment. Stratification of patients Following the overall determination of trametinib efficacy against OSCC, we turned our attention to therapeutic outcome of OSCC patients. We began by reexamining our results by sex, tumor location, age of the dog, and tumor initial volume ( Table 4 and Figure 6 ). In no case did the difference in treatment outcomes between various groupings reach statistical significance. We next considered the histological and molecular phenotype of the tumors. As shown in Figure 7A , no significant difference was present in the response of tumors according to their histological subtype. We then turned to molecular signatures. Since we knew that BRAF mutations were present in at least some tumors, we conducted BRAF genotyping on each tumor. Pretreatment tumor biopsy samples were digested to isolate DNA, and BRAF was amplified via PCR. Sanger sequencing was used to determine the BRAF sequence. The tumor samples were heterogeneous, but any detectable level of the target gene mutation (thymine to adenine at the indicated position, Figure 7B ) was considered sufficient to classify the tumor as BRAF mutant positive. Comparison of the tumor volume changes demonstrated a statistically significant difference (p = 0.01) between treatment outcomes for BRAF wildtype (WT) versus mutant (p.V595E) tumors, with the BRAF p.V595E expressing tumors shrinking to a greater degree, and with greater frequency, than BRAF WT tumors ( Figure 7C ). Discussion Our study demonstrates that trametinib, given orally at home daily, can have a substantial effect in reducing the size of ~ 30% of OSCC tumors in dogs. Only a single case so far resulted in a complete response, with no tumor remaining visible by CT. Two tumors did have nearly complete responses however, with > 90% reduction in tumor volume, and it is possible that a complete response might have occurred following a longer treatment duration. Thus, we currently consider trametinib treatment to be a neoadjuvant intervention, which might help to reduce the size of some tumors, allowing them to be better surgical candidates. As shown in Table 2 and Fig. 6 , patient sex, tumor location, patient age, and initial tumor size all failed to predict drug efficacy. However, BRAF mutational status was predictive, with canines having BRAF mutant tumors showing significantly better response to drug than canines with BRAF wildtype tumors (p = 0.01). The ORR for BRAF WT tumors was only 38%, compared to 86% for BRAF p.V595E tumors. Moreover, of the tumors that responded to drug, BRAF WT tumors saw only an average of 7% reduction in volume (median change 3% volume increase), while BRAF p.V595E tumors saw an average volume reduction of 71% (median change 89% volume reduction). In humans trametinib is typically prescribed for BRAF -mutant tumors, alongside the BRAF inhibitor dabrafenib 34 , 40 – 42 . In dogs, we have evidence that BRAF mutation may drive some OSCC tumor growth, but do not know what activating mutations or signaling pathways drive other OSCCs 10 . It is unclear to us if inhibiting MEK might only be a viable strategy when BRAF has been mutated, with other driving proteins somehow bypassing MEK activity, or if perhaps other driving proteins cause drug export proteins which target trametinib to be overexpressed. Screening for BRAF mutation can be accomplished via comparatively simple PCR amplification and Sanger sequencing, and we hope to see the Cornell AHDC offer BRAF screening as a service in the near future. When we designed this study, we started with a ‘do no harm’ approach. In our experience, following a patient’s first arrival to the clinic, it takes approximately two weeks before surgery (i.e., the current standard of care) can be scheduled and conducted. Our current approach for enrolled patients has been to immediately prescribe trametinib. This is typically delivered to the owners in 1–3 days, leading to roughly 12 days of drug treatment before the dog’s next examination. Notably, dogs were removed from the trial immediately if progressive disease was detected following any scheduled examination. Thus, only patients showing responsive disease were kept on the drug for the full 8-week duration. Moreover, the treatment is relatively convenient, as it can be administered at home, and so does not require routine hospital visits. It is also well tolerated: while several grade 1 and grade 2 adverse events were detected in blood chemistry panels, they were inconsistent and we consider it unlikely that they were a result of trametinib treatment. The two grade 3 events (ALP levels in dogs #4 and #20) were preceded by grade 2 elevations in ALP (both dogs) and ALT (dog #20) which were present during the initial bloodwork, suggesting liver damage may have been in place before drug was given, and making it unlikely that the drug was responsible for the grade 3 events. Some grade 1 gastrointestinal distress was reported by owners but was managed with minor additional care. Combined, this suggests that trametinib is a low-risk addition to the current standard of care for canine OSCC. Ethical concerns prevented our ability to use an untreated control group. CT scans to accurately assess tumor volume require anesthesia, which is a procedure with some inherent risk and expense 43 , and we could not ethically justify subjecting dogs to full-body anesthesia and radiation exposure simply to gather numbers on exact tumor growth rates absent drug. These concerns were magnified by the comparatively small number of dogs we were able to recruit into our study, and the reality that every control patient would have removed a treated patient from our already limited pool. Thus, we cannot make any arguments regarding whether treated tumors which did grow grew slower than they might have been expected to do otherwise. However, experience has taught us that tumors do not shrink on their own, and that dogs rarely if ever show a placebo effect after drug treatment, and so we are confident in our overall results regarding tumor drug response. A small number of clinical trials examining canine OSCC have been conducted in the past 25 years. One early study examined photodynamic therapy with 2-(1-hexyloxyethyl)-2-devinylpyrooheophorbide-a (HPPH). The authors reported that they examined eleven dogs, and remaining tumor was detected in 9 of the 11 dogs (82%), with two dogs having a complete response (18%). However, following surgical intervention, eight dogs (73%) had no tumor recurrence after 17 months. The authors reported that it took up to 6 weeks to heal from treatment, that the treatment could only be provided in the hospital over several days, and that tooth loss occurred at the site of treatment 44 . A more recent investigation examined hyper-fractionated radiation combined with surgery, and concluded that surgery was still the gold standard treatment, but that postoperative radiation could extend survival times in cases where clean margins could not be obtained 45 . A water-soluble form of paclitaxel (trade name Paccal Vet) was used in experiments treating 32 dogs with a variety of tumors, including 3 with squamous cell carcinomas 46 . The drug was effective in these dogs (CR or PR), but the majority of dogs in the study were reported to experience grade 1 to grade 4 adverse events which were attributed to the treatment. The drug initially had conditional approval by the FDA, but that approval has since been revoked. Finally, a series of studies has investigated the non-steroidal, anti-inflammatory compound piroxicam either alone 47 , or with cisplatin 48 or carboplatin 49 . When treated with piroxicam alone, three out of seventeen dogs (18%) responded to the drug, with an additional five dogs (29%) reaching a stable disease state. In combination with cisplatin, 5 of 9 dogs (56%) showed signs of remission, although all dogs had substantial but manageable toxicity, particularly renal toxicosis. When given with carboplatin, 4 of 7 dogs (57%) showed a complete response, with 2 of 7 (29%) showing a partial response. However, mild-to-moderate gastrointestinal side effects were noted, albeit none requiring hospitalization. By comparison, trametinib treatment has a PR or CR response rate of 30%, which increases to 71% when only BRAF mutant-positive tumors are considered. The drug had no notable toxic effects, and the entire treatment could be completed at home. Interestingly, our results closely mirror similar results in a human clinical trial examining mandibular ameloblastoma 50 , which resulted in a mix of partial and near-complete drug responses, once again showing the incredible power of dogs as translational models of disease and disease treatment. While we are very excited about these results, there are several key limitations to this study. Many of our key conclusions are drawn from CT measurements. These are inherently limited by occasional unclean margins in some tumors, and the reality that tumor volumes identified by CT are likely a combination of tumor tissue and surrounding inflammation, both of which impact the accuracy of the measurements. Moreover, we do not yet have longitudinal data to determine tumor recurrence, and in fact many patients were treated with surgery immediately following the conclusion of drug treatment to prevent recurrence to the greatest degree possible. While we were able to treat narrower margins, this does mean that our long-term survival data will never be directly comparable to some other studies. Because the drug treatment was given at home, our data may reflect dogs whose owners did not follow the dosing regimen correctly. Similarly, we do not account for confounding elements (e.g., dogs living in a home in which tobacco is routinely smoked, or drug doses being given at different times of day) which might have affected tumor growth, drug efficacy, or overall dog health. However, this does mean our trial reflects real-world conditions better than if all animals had been hospitalized for the duration of treatment. Further, as is common in veterinary clinical trials, we were only able to enroll a comparatively small number of dogs to the study. This is particularly a concern for BRAF -based stratification. While there was a statistically significant difference in outcomes between patient populations, we would feel much more confident in this as a single-factor determinant of efficacy if we had been able to study a greater number of dogs with BRAF -mutant tumors. We are seeking to address this shortcoming in the near future, and we do currently feel confident in this approach as a safe, affordable, and efficacious neoadjuvant therapy which can help shrink OSCC tumors in ~ 33% of dogs prior to surgical intervention. Conclusions The MEK inhibitor trametinib is a safe, effective way to reduce the size of oral squamous cell carcinoma tumors in dogs. While treating dogs without consideration for their characteristics results in ~ 30% of patients seeing a good drug response, it is likely that treating only dogs with BRAF -mutant tumors will raise that rate of response to at least 70%. The drug is orally available, can be given to dogs by the owner at home, and results can be seen in as little as 2 weeks of treatment. Declarations Author Contributions WPK and SP designed the study and wrote the manuscript. Dental diplomates SP, NF, and ALW worked with residents EC, MED, and CW to conduct most patient interactions. Oncologists CEB, KRH, and SRS oversaw all drug treatment decisions. ALK acted as clinical trial coordinator. MB conducted all BRAF screening, and pathologist GED conducted all histology. Radiologist ABT-D conducted and interpreted all CT scanning. WPK and PCC conducted all statistical analyses. Acknowledgements We would like to thank all the staff at CUHA, and all the owners and dogs who participated in this study, without whom these advances would not have been possible. This material is based upon work supported by the Cornell Richard P. Riney Canine Health Center Research Grants Program, a grant made available to the College of Veterinary Medicine, Cornell University. The authors declare no conflicts of interest pertaining to this study. Ethics Statement All animal procedures were reviewed and approved by Cornell University’s Institutional Animal Care and Use Committee (protocol #2023-0034). Informed signed consent was obtained from all dog owners prior to enrollment. References Cray, M., Selmic, L. E. & Ruple, A. Demographics of dogs and cats with oral tumors presenting to teaching hospitals: 1996–2017. J Vet Sci 21 (2020). https://doi.org/10.4142/jvs.2020.21.e70 Fulton, A. J., Nemec, A., Murphy, B. G., Kass, P. H. & Verstraete, F. J. M. Risk factors associated with survival in dogs with nontonsillar oral squamous cell carcinoma 31 cases (1990–2010). Journal of the American Veterinary Medical Association 243, 696–702 (2013). https://doi.org/10.2460/javma.243.5.696 Wingo, K. 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Signal Transduction and Targeted Therapy 6, 386 (2021). https://doi.org/10.1038/s41392-021-00780-4 Murugan, A. K., Munirajan, A. K. & Tsuchida, N. Ras oncogenes in oral cancer: The past 20 years. Oral Oncology 48, 383–392 (2012). https://doi.org/10.1016/j.oraloncology.2011.12.006 Punekar, S. R., Velcheti, V., Neel, B. G. & Wong, K.-K. The current state of the art and future trends in RAS-targeted cancer therapies. Nature Reviews Clinical Oncology 19, 637–655 (2022). https://doi.org/10.1038/s41571-022-00671-9 Pylayeva-Gupta, Y., Grabocka, E. & Bar-Sagi, D. RAS oncogenes: weaving a tumorigenic web. Nature Reviews Cancer 11, 761–774 (2011). https://doi.org/10.1038/Nrc3106 Verstraete, F. J. M. Mandibulectomy and maxillectomy. Veterinary Clinics of North America: Small Animal Practice 35, 1009–1039 (2005). https://doi.org/10.1016/j.cvsm.2005.03.005 Wright, A. L., Peralta, S. & Fiani, N. Case report: Spontaneous mandibular body regeneration following unilateral subtotal mandibulectomy in a 3-month-old French bulldog. Frontiers in Veterinary Science 10 (2023). https://doi.org/10.3389/fvets.2023.1281232 Bull, I., Ziener, M. L., Storli, S. H. & Arendt, M. L. Quality of life after partial mandibulectomy or maxillectomy in 45 dogs with oral tumors. Journal of Veterinary Dentistry 40, 329–337 (2023). https://doi.org/10.1177/08987564231164483 Fiani, N. & Peralta, S. Extended subtotal mandibulectomy for the treatment of oral tumors invading the mandibular canal in dogs-a novel surgical technique. Frontiers in veterinary science 6, 339 (2019). https://doi.org/10.3389/fvets.2019.00339 Warshaw, S. L., Carney, P. C., Peralta, S. & Fiani, N. Piezosurgical bone-cutting technology reduces risk of maxillectomy and mandibulectomy complications in dogs. Journal of the American Veterinary Medical Association 261, 1–7 (2023). https://doi.org/10.2460/javma.23.03.0130 Bar-Am, Y. & Verstraete, F. J. M. Elastic training for the prevention of mandibular drift following mandibulectomy in dogs: 18 cases (2005–2008). Veterinary Surgery 39, 574–580 (2010). https://doi.org/10.1111/j.1532-950X.2010.00703.x Katt, W. P. et al. The MEK inhibitor trametinib is effective in inhibiting the growth of canine oral squamous cell carcinoma. Scientific Reports 15, 7069 (2025). https://doi.org/10.1038/s41598-025-90574-3 Bartel, A. et al. Expression of mutated BRAFV595E kinase in canine carcinomas—an immunohistochemical study. Veterinary Sciences 11 (2024). Bahar, M. E., Kim, H. J. & Kim, D. R. Targeting the RAS/RAF/MAPK pathway for cancer therapy: from mechanism to clinical studies. Signal Transduction and Targeted Therapy 8, 455 (2023). https://doi.org/10.1038/s41392-023-01705-z Song, Y. et al. Targeting RAS–RAF–MEK–ERK signaling pathway in human cancer: Current status in clinical trials. Genes & Diseases 10, 76–88 (2023). https://doi.org/10.1016/j.gendis.2022.05.006 Rubinson, D. A. et al. Sotorasib Is a pan-RASG12C inhibitor capable of driving clinical response in NRASG12C cancers. Cancer Discovery 14, 727–736 (2024). https://doi.org/10.1158/2159-8290.CD-23-1138 Wright, C. J. M. & McCormack, P. L. Trametinib: first global approval. Drugs 73, 1245–1254 (2013). https://doi.org/10.1007/s40265-013-0096-1 Infante, J. R. et al. Safety, pharmacokinetic, pharmacodynamic, and efficacy data for the oral MEK inhibitor trametinib: a phase 1 dose-escalation trial. The Lancet Oncology 13, 773–781 (2012). https://doi.org/10.1016/S1470-2045(12)70270-X Khan, Z. M. et al. Structural basis for the action of the drug trametinib at KSR-bound MEK. Nature 588, 509–514 (2020). https://doi.org/10.1038/s41586-020-2760-4 Takada, M. et al. Population pharmacokinetics, pharmacodynamics and safety properties of trametinib in dogs with cancer: A phase I dose escalating clinical trial. Veterinary and Comparative Oncology 22, 410–421 (2024). https://doi.org/10.1111/vco.12989 Odogwu, L. et al. FDA approval summary: Dabrafenib and trametinib for the treatment of metastatic non-small cell lung cancers harboring BRAF V600E mutations. Oncologist 23, 740–745 (2018). https://doi.org/10.1634/theoncologist.2017-0642 Leonowens, C. et al. Concomitant oral and intravenous pharmacokinetics of trametinib, a MEK inhibitor, in subjects with solid tumours. British Journal of Clinical Pharmacology 78, 524–532 (2014). https://doi.org/10.1111/bcp.12373 Peralta, S. et al. Comparative transcriptional profiling of canine acanthomatous ameloblastoma and homology with human ameloblastoma. Scientific Reports 11, 17792 (2021). https://doi.org/10.1038/s41598-021-97430-0 Nemec, A., Murphy, B., Kass, P. H. & Verstraete, F. J. M. Histological subtypes of oral non-tonsillar squamous cell carcinoma in dogs. Journal of Comparative Pathology 147, 111–120 (2012). https://doi.org/10.1016/j.jcpa.2011.11.198 Nguyen, S. M., Thamm, D. H., Vail, D. M. & London, C. A. Response evaluation criteria for solid tumours in dogs (v1.0): a Veterinary Cooperative Oncology Group (VCOG) consensus document. Veterinary and Comparative Oncology 13, 176–183 (2015). https://doi.org/10.1111/vco.12032 LeBlanc, A. K. et al. Veterinary Cooperative Oncology Group—Common Terminology Criteria for Adverse Events (VCOG-CTCAE v2) following investigational therapy in dogs and cats. Veterinary and Comparative Oncology 19, 311–352 (2021). https://doi.org/10.1111/vco.12677 Wang, W. et al. Phase I/II trial of concurrent extracranial palliative radiation therapy with Dabrafenib and Trametinib in metastatic BRAF V600E/K mutation-positive cutaneous Melanoma. Clinical and Translational Radiation Oncology 30, 95–99 (2021). https://doi.org/10.1016/j.ctro.2021.08.006 Lugowska, I., Kosela-Paterczyk, H., Kozak, K. & Rutkowski, P. Trametinib: a MEK inhibitor for management of metastatic melanoma. Onco Targets Ther 8, 2251–2259 (2015). https://doi.org/10.2147/Ott.S72951 Corcoran, R. B. et al. Combined BRAF and MEK inhibition with dabrafenib and trametinib in BRAF V600–mutant colorectal cancer. Journal of Clinical Oncology 33, 4023–4031 (2015). https://doi.org/10.1200/JCO.2015.63.2471 Gruenheid, M. et al. Risk of anesthesia-related complications in brachycephalic dogs. Journal of the American Veterinary Medical Association 253, 301–306 (2018). https://doi.org/10.2460/javma.253.3.301 McCaw, D. L. et al. Treatment of canine oral squamous cell carcinomas with photodynamic therapy. British Journal of Cancer 82, 1297–1299 (2000). https://doi.org/10.1054/bjoc.1999.1094 Riggs, J. et al. Outcomes following surgical excision or surgical excision combined with adjunctive, hypofractionated radiotherapy in dogs with oral squamous cell carcinoma or fibrosarcoma. Journal of the American Veterinary Medical Association 253, 73–83 (2018). https://doi.org/10.2460/javma.253.1.73 von Euler, H., Rivera, P., Nyman, H., Häggström, J. & Borgå, O. A dose-finding study with a novel water-soluble formulation of paclitaxel for the treatment of malignant high-grade solid tumours in dogs. Veterinary and Comparative Oncology 11, 243–255 (2013). https://doi.org/10.1111/j.1476-5829.2011.00314.x Schmidt, B. R., Glickman, N. W., DeNicola, D. B., Gortari, A. E. d. & Knapp, D. W. Evaluation of piroxicam for the treatment of oral squamous cell carcinoma in dogs. Journal of the American Veterinary Medical Association 218, 1783–1786 (2001). https://doi.org/10.2460/javma.2001.218.1783 Boria, P. A. et al. Evaluation of cisplatin combined with piroxicam for the treatment of oral malignant melanoma and oral squamous cell carcinoma in dogs. Journal of the American Veterinary Medical Association 224, 388–394 (2004). https://doi.org/10.2460/javma.2004.224.388 De Vos, J. P. et al. Piroxicam and carboplatin as a combination treatment of canine oral non-tonsillar squamous cell carcinoma: a pilot study and a literature review of a canine model of human head and neck squamous cell carcinoma. Veterinary and Comparative Oncology 3, 16–24 (2005). https://doi.org/10.1111/j.1476-5810.2005.00065.x Grynberg, S. et al. Neoadjuvant BRAF-targeted therapy for ameloblastoma of the mandible: an organ preservation approach. JNCI: Journal of the National Cancer Institute , djad232 (2023). https://doi.org/10.1093/jnci/djad232 Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx SupplementalinformationDocument1.docx Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Jul, 2025 Reviews received at journal 18 Jul, 2025 Reviews received at journal 17 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviews received at journal 30 Jun, 2025 Reviewers agreed at journal 24 Jun, 2025 Reviewers invited by journal 12 Jun, 2025 Submission checks completed at journal 11 Jun, 2025 First submitted to journal 10 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6821917","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":470590543,"identity":"1322be7f-64d2-4690-b07b-9cf436bd9f85","order_by":0,"name":"William P. Katt","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"P.","lastName":"Katt","suffix":""},{"id":470590544,"identity":"1fe4c1a0-f4f8-4473-ac39-460fc2029dbb","order_by":1,"name":"Cheryl E. Balkman","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Cheryl","middleName":"E.","lastName":"Balkman","suffix":""},{"id":470590548,"identity":"c1f8a01f-0012-4b5c-9501-ba1f667f54c8","order_by":2,"name":"Michael Byron","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Byron","suffix":""},{"id":470590550,"identity":"7be21089-9a26-458e-ad9f-3f03dfbb3cc3","order_by":3,"name":"Patrick C. Carney","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"C.","lastName":"Carney","suffix":""},{"id":470590551,"identity":"3d52d576-5c54-47c2-8fc6-519851d393b4","order_by":4,"name":"Matthew E. Drozd","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"E.","lastName":"Drozd","suffix":""},{"id":470590552,"identity":"2bf8dbf5-b101-4ac5-8e40-e044dc29f6bd","order_by":5,"name":"Gerald E. Duhamel","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Gerald","middleName":"E.","lastName":"Duhamel","suffix":""},{"id":470590553,"identity":"9c9ac4a8-426b-460b-bc95-d7051c7b7998","order_by":6,"name":"Nadine Fiani","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Nadine","middleName":"","lastName":"Fiani","suffix":""},{"id":470590554,"identity":"8414857b-0dd4-4db2-a4e8-f22fddd5c0e3","order_by":7,"name":"Kelly R. Hume","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Kelly","middleName":"R.","lastName":"Hume","suffix":""},{"id":470590555,"identity":"ae48ecd7-5c64-4a64-a93a-036e19ee3dfc","order_by":8,"name":"Andrea L. King","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"L.","lastName":"King","suffix":""},{"id":470590556,"identity":"d3b8e73f-c73a-4d93-9efb-3c5e9bd8e168","order_by":9,"name":"Skylar R. Sylvester","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Skylar","middleName":"R.","lastName":"Sylvester","suffix":""},{"id":470590557,"identity":"6199a6be-5c31-40bf-bdb3-72ca74d3b153","order_by":10,"name":"Amy B. Todd-Donato","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Amy","middleName":"B.","lastName":"Todd-Donato","suffix":""},{"id":470590558,"identity":"01c84fd8-16ae-47b0-b975-303b06ad624d","order_by":11,"name":"Cailin E. Winokur","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Cailin","middleName":"E.","lastName":"Winokur","suffix":""},{"id":470590559,"identity":"e01decef-080b-4a0f-a5b8-7480a928ccea","order_by":12,"name":"Alexandra L. Wright","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"L.","lastName":"Wright","suffix":""},{"id":470590560,"identity":"0b7b59ae-16b4-4292-80ea-5619f2f31b79","order_by":13,"name":"Santiago Peralta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYFAC5oMPPlSAWYwHHgCJBsJa2JINZ5yBMA8kEKeFx0yat40ULfKz25INZ86zkzOf3XwAqMVGdsMBAloM7hw++ODjtmRjmTvHEoBa0owJa5FIA9qy7UDiDIkcA6CWw4kEtcjPyAH6ZQ5IS/4HoJb/hLUw3ABpaQDbAvL+AcJaDO4cAwbysWRjCYk0oMMMko1nEnTY7GZgVNbYyUlIJD8ExqmdbB9Bh0mgWkpIOaaWUTAKRsEoGAVYAACjcEuBlysofQAAAABJRU5ErkJggg==","orcid":"","institution":"Cornell University","correspondingAuthor":true,"prefix":"","firstName":"Santiago","middleName":"","lastName":"Peralta","suffix":""}],"badges":[],"createdAt":"2025-06-04 15:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6821917/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6821917/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84809796,"identity":"83992b51-ea04-41bf-8a25-b71176ce6338","added_by":"auto","created_at":"2025-06-17 14:44:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":584608,"visible":true,"origin":"","legend":"\u003cp\u003eSignaling in COSCC. A) Gene set enrichment analysis was conducted on 8 OSCC samples and 5 healthy gingiva samples. The enrichment score graph illustrates upregulation of most genes contained in the KRAS.600_UP.V1_UP gene list (defined as “Genes upregulated in four lineages of epithelial cell lines over-expressing an oncogenic form of KRAS [GeneID=3845] gene”). B) RAS-signal transduction is typically initiated by extracellular signaling molecules binding to G-protein coupled receptors or other receptor tyrosine kinases. RAS is then activated, and initiates a signaling cascade from RAF, to MEK, to ERK, which initiates gene transcription leading to cell growth. Targeted small molecules have been described which target most proteins in this signaling pathway. C) Three-dimensional rendered computed tomographic image following rostral maxillectomy in a dog with papillary OSCC. The arrow indicates the excised portion and illustrates how large anatomical areas are impacted by surgical standard of care. D) Radiographic image of the excised surgical specimen from the same case shown in panel C; the arrows indicate areas of osseous tumor invasion. E) The small molecule trametinib is an FDA-approved inhibitor of MEK.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/eaf6ca31afb09cd9b4199126.png"},{"id":84809797,"identity":"ce88eb8c-11ad-4160-ba9a-909a586537c8","added_by":"auto","created_at":"2025-06-17 14:44:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1455793,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative photomicrographs of patient tumors (10X magnification) highlighting invading neoplastic epithelial cell islands (arrows) interspersed with tumor-associated fibrovascular stroma (stars). A-B) H\u0026amp;E and Ki67 staining of dog #4 conventional OSCC with accumulation of compact laminated keratin (keratin pearls; indicated by white wedges). C-D) H\u0026amp;E and Ki67 staining of dog #8 papillary OSCC. E-F) H\u0026amp;E and Ki67 staining of dog #12 basaloid OSCC.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/e9bfbf7547b18aa25abb16a7.png"},{"id":84809798,"identity":"d1c162e3-f073-44cd-a27f-8447543e0138","added_by":"auto","created_at":"2025-06-17 14:44:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":415207,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of trametinib of OSCC tumor volume. A) Canine patients with OSCC were administered trametinib (between 0.015 mg/kg and 0.035 mg/kg) daily, and tumors were measured every two weeks. Their tumors were measured before treatment began, and at the conclusion of treatment, using CT scanning. Patients are displayed in order of enrollment. B) The change in volume of each patient’s tumor was calculated ((exit volume – initial volume)/(initial volume)*100).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/3f4041cf0ab9908e125394c0.png"},{"id":84810940,"identity":"4aff86ce-bf31-4100-8b9f-92df6e86e23c","added_by":"auto","created_at":"2025-06-17 14:52:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":841091,"visible":true,"origin":"","legend":"\u003cp\u003eExamples of successful trametinib treatment. A) Tumor volume changes for four dogs with PR or CR following trametinib treatment. Patient tumor sizes were tracked with calipers at each in-person visit to the clinic. Tumor volume was estimated based on measurement in three dimensions. B) Photograph of patient #9’s tumor before trametinib treatment. C) Patient #16’s tumor before trametinib treatment. D) Photograph of patient #9’s tumor on the day it exited from the study. E) Patient #16’s tumor on the day it exited from the study. These photos show that the visible portions of some tumors were difficult or impossible to identify by the end of treatment.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/e84f59fd2dadab992ae71690.png"},{"id":84810942,"identity":"66e86c0e-1d48-438e-a8e9-c7d5b6022f7f","added_by":"auto","created_at":"2025-06-17 14:52:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":770542,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative CT images of OSCC tumors at the three imaging timepoints for study dog #9 (A, B, E, F, I, J) and study dog #14 (C, D, G, H, K, L) showing decreasing tumor size and bone healing over 56 days of treatment. A) Initial post-contrast soft tissue window CT image of dog #9. B) Initial bone window CT image of dog #9. C) Initial post-contrast soft tissue window CT image of dog #14. D) Initial bone window CT image of dog #14 after Day 28. E) Post-contrast soft tissue window CT image of dog #9 after Day 28. F) Bone window CT image of dog #9 after Day 28. G) Post-contrast soft tissue window CT image of dog #14 after Day 28. H) Bone window CT image of dog #14 after Day 28. I) Post-contrast soft tissue window CT image of dog #9 after Day 56. J) Bone window CT image of dog #9 after Day 56. K) Post-contrast soft tissue window CT image of dog #14 after Day 56. L) Bone window CT image of dog #14 after Day 56. In each image, a white arrow indicates the location of the original soft tissue (ST) window/tumor location for the dog. Window width/ window level settings for the images are: bone = 1100/4500 and soft tissue = 90/320.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/8eca42d7a6aba7ca0bcfe80c.png"},{"id":84810944,"identity":"ecb52bb0-5e8b-4278-a9e6-28563f874113","added_by":"auto","created_at":"2025-06-17 14:52:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":583045,"visible":true,"origin":"","legend":"\u003cp\u003eDrug effect based on gross physical and patient demographic parameters. Patients were considered by A) sex (MC = male castrated, FS = female spayed), B) tumor location, C) age, or D) initial tumor volume. In no case was a statistically significant difference detected in the drug response between groups. p-values between groups were calculated with the Mann-Whitney U test (A), or Kruskal-Wallis test (B, C, and D), and rounded to the nearest 0.01.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/715de8e38700524665c6e21f.png"},{"id":84809808,"identity":"26c13d98-fceb-404f-bfce-b93d04979299","added_by":"auto","created_at":"2025-06-17 14:44:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":393867,"visible":true,"origin":"","legend":"\u003cp\u003eBRAF mutational status predicts trametinib efficacy. A) B) Tumors were considered to harbor a mutation in \u003cem\u003eBRAF\u003c/em\u003e if a detectable adenine signal was observed overlapping the expected thymine at the * marked position, thus demonstrating that the \u003cem\u003eBRAF\u003c/em\u003e p.V595E mutant would be encoded. C) Separation of tumor volume changes by \u003cem\u003eBRAF\u003c/em\u003e mutational status shows a statistically significant difference in effect, with \u003cem\u003eBRAF\u003c/em\u003emutant tumors shrinking much more than \u003cem\u003eBRAF\u003c/em\u003e wildtype (WT) tumors. p-values between groups were calculated with the Kruskal-Wallis test (A), or Mann-Whitney U test (C), and rounded to the nearest 0.01.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/56d2b64e8f4e77216910aec5.png"},{"id":84813911,"identity":"cd960434-6df8-401f-8755-138062c56733","added_by":"auto","created_at":"2025-06-17 15:16:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5726941,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/35dde1ce-8662-4b92-9582-adf5a5f38aa9.pdf"},{"id":84809795,"identity":"96917ddd-99b6-4b59-97f6-1b5227780177","added_by":"auto","created_at":"2025-06-17 14:44:25","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13582,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/d08a3cbcc214d51ac3a6bb94.xlsx"},{"id":84809806,"identity":"3c174be9-f35d-4f30-a4bb-721a05336219","added_by":"auto","created_at":"2025-06-17 14:44:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":694749,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalinformationDocument1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/2aabd35930b8cf51cba08285.docx"},{"id":84810938,"identity":"7c418666-9e85-4523-9efc-2f94aa369a15","added_by":"auto","created_at":"2025-06-17 14:52:26","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":66202,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6821917/v1/398eebae89eb347b0714bab9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clinical application of the MEK inhibitor trametinib in dogs with oral squamous cell carcinoma","fulltext":[{"header":"Introduction","content":" \u003cp\u003eOral tumors in dogs are relatively common, affecting an estimated 0.5% of dogs in their lifetimes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and representing 6% of the total cancer burden in dogs\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. One of the most common oral canine tumors is oral squamous cell carcinoma (OSCC), an aggressive and rapidly growing disease of epithelial origin that makes up ~\u0026thinsp;19% of all oral tumors in dogs\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Ki67 staining of tumor sections shows relatively high proliferative rate compared to other common oral tumors such as canine acanthomatous ameloblastoma (CAA)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. OSCC is also metastatic, with up to 20% of dogs showing signs of metastasis at the time of diagnosis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. OSCC in dogs is locally invasive and grows in multiple anatomical planes, which can cause severe pain and dysfunction, and in some cases leads to more severe morbidity including pathological fracture\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the last decade, canine OSCC has been the focus of significant research which has helped to define the molecular landscape of the disease. Crucially, recent findings have shown that OSCC has highly activated RAS signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA)\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. RAS is a small GTPase which is activated by extracellular signaling through transmembrane receptors, and its activation initiates multiple signaling cascades supporting cell growth and survival\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, the most important of these in the context of the sustained growth necessary to support a tumor is the RAS-RAF-MEK-ERK pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDespite recent advances in understanding the molecular mechanisms driving OSCC, the approach to treatment in canine patients has not yet seen any benefit. The standard of care is largely centered around wide-margin surgical excision of the tumor (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), with radiotherapy being occasionally employed to attempt to shrink or halt the growth of a tumor which is not surgically treatable, or as an adjuvant treatment to surgery\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. While this surgical approach is highly effective, with very low recurrence rates when tumors are completely excised before becoming metastatic, the treatment is also highly invasive, invariably resulting in facial disfiguration and leading to difficulty eating in ~\u0026thinsp;30% of dogs\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Moreover, larger surgeries lead to both longer healing times and greater chances for postoperative complications and pain\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These realities led us to investigate whether the RAS pathway could be targeted to shrink OSCC tumors.\u003c/p\u003e \u003cp\u003eWhile gene set enrichment analysis clearly indicated the upregulation of RAS signaling in OSCC\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, it is currently unclear which mutations lead to this activation. RAS itself does not appear to be mutated in most OSCC, despite being frequently mutated in CAA\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The RAS signaling partner BRAF is mutated (V595E, corresponding to the constitutively activating V600E mutation in humans), but only in about a third of OSCCs, predominantly those of the papillary subtype\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Because it is unclear if a single mutated protein is common to all, or even most, OSCC in dogs, when we set out to find a targeted therapeutic approach to OSCC we prioritized drugs which worked on wildtype proteins and did not require a mutation to bind. This ruled out the promising new RAS inhibitors such as sotorasib, which only tightly bind to mutant RAS proteins, but left several drugs available, including the MEK inhibitor trametinib\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTrametinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) is a small molecule which inhibits MEK1 and MEK2\u003csup\u003e30,31\u003c/sup\u003e. It binds to the non-phosphorylated version of either protein, blocking their catalytic activity and thus their ability to activate ERK. The drug occupies an allosteric pocket close to the ATP binding pocket on MEK, and crystal structures show that it not only directly blocks binding of BRAF to MEK, thus preventing MEK phosphorylation, but also seems to stabilize binding of MEK to KSR, the kinase suppressor of RAS, which presumably acts to further reduce the binding affinity of MEK for BRAF\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Trametinib has been extensively studied, with a well verified safety profile\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and it is approved in the US by the FDA for the treatment of BRAF-mutant melanoma or non-small cell lung cancers in humans, in which case it is used alongside the BRAF inhibitor dabrafenib\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Further, trametinib has high oral bioavailability in both dogs\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and humans\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, so can be taken at home. We have shown that it is effective at reducing the growth of canine OSCC cell culture models and murine xenograft models\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe objective of this study was to test the hypothesis that daily treatment with trametinib can shrink OSCC tumors in domestic dogs while being well tolerated at effective doses. Dogs with spontaneously occurring OSCC were treated for 2\u0026ndash;8 weeks with trametinib. Tumor volumes were determined by contrast-enhanced computed tomographic (CT) scanning before and periodically during treatment. We found that 50% of dogs experienced a decrease in tumor volume, or had the tumor stop growing, over the course of treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe study design was reviewed and approved by Cornell University\u0026rsquo;s Institutional Animal Care and Use Committee, protocol number 2023-0034. Study design and approval was in place before animal recruitment began. Dogs were eligible for enrollment if they had an OSCC, as confirmed histologically by the Animal Health Diagnostic Center (AHDC) at Cornell University, arising from a mucosal surface, were able to undergo at least two rounds of general anesthesia, and could visit the clinic at least every two to four weeks during the study period. Dogs were ineligible if they were pregnant or lactating, their oral tumors had been previously excised, they had previously received radiation or chemotherapy, they were less than one year old, or they had signs of metastatic disease or of any unrelated chronic debilitating illness. Exclusion diseases included diabetes mellitus, uncontrolled Cushing\u0026rsquo;s or Addison\u0026rsquo;s disease, renal insufficiency, congestive heart failure, and any other cancer. Following enrollment (day 0), oral trametinib was immediately prescribed (0.015 mg/kg to 0.035 mg/kg once daily, with dose escalating between dogs when three prior patients had been given a lower dose and found to tolerate it with no adverse effects, as detailed below). Trametinib was sourced from the Best Pet RX Pharmacy as a formulation including dimethyl sulfoxide and was given to the dog by the owners at home. Owners were given information concerning drug handling and use, and a list of common side effects along with a questionnaire to document any adverse events from drug treatment.\u003c/p\u003e \u003cp\u003eDrug response and tumor progress were monitored during biweekly physical examinations of each dog. These examinations included tumor measurement with calipers, a complete blood count, and a serum biochemistry panel. Caliper measurements were conducted in three planes whenever possible, and in two planes when a third plane could not be reasonably identified. Full tumor staging was conducted on days 0, ~\u0026thinsp;28, and ~\u0026thinsp;56. Staging included a cytological and/or histological assessment of regional lymph nodes to check for metastasis, and a contrast-enhanced (2mL/kg iohexol, 350 mg/mL iodine) CT head exam, using a 16-slice Aquilion LB helical CT (Canon Medical Systems USA, Inc., Tustin, CA) to assess the main tumor burden. The head CT examination included general anesthesia using standard-of-care individualized protocols determined by a board-certified veterinary anesthesiologist, and was conducted by acquiring continuous axial slices, with a slice thickness of 0.5 mm to 2.0 mm and volumetric reconstructions with effective slice thicknesses of 0.3 mm to 1.0 mm. The CT imaging was analyzed by a board-certified veterinary radiologist as DICOM studies in the hospital\u0026rsquo;s Picture Archiving and Communication System (Carestream VuePACS, Rochester, NY, USA) using multiplanar reconstructions in bone and soft tissue windows, pre- and post-contrast. The tumor volume was determined by measuring the cross-sectional area of the abnormal/tumor tissue in each slice and multiplying by the slice thickness.\u003c/p\u003e \u003cp\u003eEach 2-week examination was used as a decision point for continuing trametinib treatment. If progressive disease was noted, treatment was discontinued, and the dog was shifted to the normal standard of care, as determined by the attending veterinarian. Guidelines for managing side effects were developed; mild adverse events were to be managed symptomatically, while moderate or severe problems would result in decreasing the drug dose or discontinuing the study at the discretion of the attending veterinarian. The experimental unit in all reported data is a single dog being treated, and only domestic companion animals with naturally occurring OSCC were used in this study. No experimental animals were used to supplement enrollment numbers. All dogs received treatment, no negative control group was included due to anticipated enrollment numbers and general understanding of the growth rate of untreated tumors. The total experimental cohort was twenty dogs. Because no negative control was included, it was impossible to blind study participants as to the nature of treatment. However, all data was analyzed by an investigator unaware of which dog\u0026rsquo;s data they were working on, then compiled by another investigator who could match the data to the dog. No efforts were made to ensure gender balance or a balance between tumor origin sites.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDose escalation\u003c/h2\u003e \u003cp\u003eThe starting dosage was 0.015 mg/kg, and dosages were escalated in increments of 0.005 mg/kg in cohorts of at least three dogs. Toxicity was monitored with complete blood counts and chemistry panels every 2 weeks. Gastrointestinal toxicity was assessed from medical histories reported by owners. If none of the dogs treated in a given cohort experienced a severe toxicity (Grade 3 or higher adverse event), the dosage for the next cohort was escalated. Cohort size varied in order to accumulate necessary safety data before escalating the dose in a new cohort. If one dog experienced severe toxicity, at least three additional dogs were treated at that dosage. If no additional dogs experienced severe toxicity at that dosage, then the escalation continued. If\u0026thinsp;\u0026ge;\u0026thinsp;2 dogs in a cohort experienced severe toxicity, additional enrollment in that cohort was stopped.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eData analysis and statistical approach\u003c/h3\u003e\n\u003cp\u003eThe cohort size of 20 dogs was supported by a power calculation performed as a paired t-test using PROC POWER in SAS 9.4. The power calculation assumed tumors averaging 20\u0026ndash;40 mm in diameter, a correlation of 0.5, and an alpha of 0.5, and suggested that 14\u0026ndash;15 dogs at a minimum would be required for an adequately powered study, based on tumor size before and after treatment.\u003c/p\u003e \u003cp\u003eCT measurements were considered to be more accurate than caliper measurements and so were used when possible as the basis for determining R.E.C.I.S.T. categorization of treatment, and for all graphs showing the difference between tumor volume at start and exit of the study. All CT data reported show the exact volume determined for each dog. Because these volumes were available for dogs that completed the study, we used them rather than \u0026lsquo;longest axis\u0026rsquo; measurements to determine R.E.C.I.S.T. categorizations. Specifically, CR was a tumor that could not be detected by CT. PR was a tumor which had a volume at least 30% smaller than the original volume. SD was a tumor with less than 30% reduction or 20% increase in volume compared to the original tumor volume. And PD was a tumor which grew more than 30%. For dog #10, no exit CT scan was conducted, so PD was based on estimated volume from caliper measurements.\u003c/p\u003e \u003cp\u003eUnivariate tests based on the degree of tumor volume change over the course of treatment were conducted to analyze differences in treatment success rate between various groups. Statistically significant differences between compared groups were determined using tests appropriate for independent samples that are not normally distributed, namely the Mann-Whitney U test (for two groupings) or Kruskal-Wallis test (for three or more groupings). These calculations were completed in GraphPad Prism 10.4.2. Differences in results between groups were considered to be statistically significant at a level of p\u0026thinsp;\u0026le;\u0026thinsp;0.05. Caliper measurements were used to follow tumor size over the course of the experiment and are used for graphs showing tumor growth over time. Where CT scans are shown in the data, the shown slice is at or near the thickest part of the tumor as determined by the veterinary radiologist. All twenty dogs are included in every analysis reported.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTwenty dogs were enrolled over a period spanning from October 2023 to February 2025 after meeting the inclusion criteria. Dogs either initially presented to the Cornell University Hospital for Animals (CUHA) or were referred by their primary practitioners . All dogs were ultimately enrolled in the veterinary clinical trial following a visit to CUHA, and all reported examinations were performed at CUHA except as indicated. Dogs were diagnosed with OSCC following routine tumor staging and histological workup\u003csup\u003e4,8,36\u003c/sup\u003e (\u003cstrong\u003eFigure 2\u003c/strong\u003e). The dogs included fifteen purebreds, with the remaining being various mixed breeds (\u003cstrong\u003eTable 1\u003c/strong\u003e). The cohort included nine spayed females (45%) and eleven castrated males (55%). Ages ranged from 3 to 14 years with a median of 9 years. Eight tumors were located on the caudal maxilla, five on the rostral maxilla, four on the rostral mandible, two on the caudal mandible, and one was sublingual. Initial tumor volume, as determined by CT, ranged from 0.27 cm\u003csup\u003e3\u003c/sup\u003e to 71.62 cm\u003csup\u003e3\u003c/sup\u003e, with an average initial volume of 23.7 cm\u003csup\u003e3\u003c/sup\u003e. Histological examination showed 3 tumors to be basaloid, 6 to be papillary, and 11 to be conventional, further broken down as 5 with poor differentiation, 5 with moderate differentiation, and 1 well differentiated\u003csup\u003e10,37\u003c/sup\u003e. All twenty histological subtypes were determined by the same pathologist.\u003c/p\u003e\n\u003cp\u003eAfter histologic confirmation of OSCC and staging, dogs were prescribed trametinib, to be given orally by the owners once daily. Tumors\u0026rsquo; response to drug treatment was classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD, consistent with widely-used R.E.C.I.S.T. reporting parameters\u003csup\u003e38\u003c/sup\u003e. As shown in \u003cstrong\u003eTable 2\u003c/strong\u003e, 6 dogs exhibited CR or PR (overall response rate [ORR] 30%), while fourteen dogs exhibited SD (20%) or PD (30%) with treatment. \u003cstrong\u003eFigure 3A\u003c/strong\u003e and \u003cstrong\u003eTable S1\u003c/strong\u003e show the tumor volume before and after treatment for each patient, while \u003cstrong\u003eFigure 3B\u003c/strong\u003e shows the percentage growth or reduction in size for each patient\u0026rsquo;s tumor, calculated as (exit volume \u0026ndash; initial volume)/(initial volume)*100. One dog (#11) was removed from the trial due to obvious tumor growth before a 1-month CT scan could be conducted, and the tumor was estimated to have roughly doubled in size by visual assessment. For all other dogs, final tumor volume was determined by CT when they exited the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile our trial was designed for 8 weeks of treatment, some tumors responded to the drug within 2 weeks. Indeed, for five dogs their tumor volume had been reduced by the date of their first post-treatment evaluation (average of 45% tumor volume reduction, average of 18 days between first and second examination). For dog #8, the tumor began to regrow by the next examination. The tumors of several dogs had almost entirely disappeared after 2-6 weeks of treatment (\u003cstrong\u003eFigure 4A\u003c/strong\u003e), which in some cases was easily apparent by external visual inspection (\u003cstrong\u003eFigures 4B-4E\u003c/strong\u003e). In patients where CR or PR was observed, we examined their CT scans to determine any other anatomical results from treatment. The soft tissue scans easily demonstrated a reduction in tumor volume (\u003cstrong\u003eFigures 5A, 5C\u003c/strong\u003e), and bone window scans showed rapid bone regrowth in some (N=2) responsive patients (\u003cstrong\u003eFigures 5B, 5D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePhysiological changes due to treatment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePatient health was monitored at home by the owners and was reported via a survey to document adverse events (\u003cstrong\u003eSupplemental information, Document 1\u003c/strong\u003e). No high-grade adverse events were detected in any of the dogs or reported by clients, although ~30% of owners did report that their dogs had transient flatulence that resolved spontaneously after a few days. Also, per one dog\u0026rsquo;s owner, that dog exhibited two vomiting episodes a few days after initiating therapy, although the animal had a history of chronic vomiting so these events may or not have been related to trametinib administration. None of the dogs had to be removed from the study and none of the dosing regimens had to be modified due to observed or reported adverse events.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePatients were also examined every ~two weeks at CUHA or by the primary practitioner in consultation with CUHA clinicians. Routine bloodwork showed no significant adverse events from the drug. \u003cstrong\u003eTable 2\u003c/strong\u003e summarizes several key values, namely neutrophil count (NEU), liver enzyme expression (alanine transaminase/ALT, aspartate transaminase/AST, and alkaline phosphatase/ALP), and markers of renal function (blood urea nitrogen/BUN, creatinine). Three dogs had their pre-treatment bloodwork completed at facilities which do not routinely measure AST levels, and these are marked as not measured (n.m.). While several dogs had assorted values that fell outside the normal range, post-treatment measurements were typically no worse than pre-treatment measurements, and represented predominantly grade 1 and grade 2 adverse events, which are summarized in \u003cstrong\u003eTable 3\u003c/strong\u003e\u003csup\u003e39\u003c/sup\u003e. Several grade 1 events were only marginally outside the normal expected range. Only two grade 3 metabolic abnormalities occurred (ALP, dogs #4 and #20) and each dog had grade 2 pre-treatment elevations in ALT and/or ALP, suggesting preexisting liver pathology unrelated to trametinib treatment. BUN and creatinine levels were only marginally outside the normal range for three dogs (#14, #17, and #18). No dog showed clear signs of neutropenia before or after treatment.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStratification of patients\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the overall determination of trametinib efficacy against OSCC, we turned our attention to therapeutic outcome of OSCC patients. We began by reexamining our results by sex, tumor location, age of the dog, and tumor initial volume (\u003cstrong\u003eTable 4\u003c/strong\u003e and \u003cstrong\u003eFigure 6\u003c/strong\u003e). In no case did the difference in treatment outcomes between various groupings reach statistical significance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next considered the histological and molecular phenotype of the tumors. As shown in \u003cstrong\u003eFigure 7A\u003c/strong\u003e, no significant difference was present in the response of tumors according to their histological subtype. We then turned to molecular signatures. Since we knew that \u003cem\u003eBRAF\u0026nbsp;\u003c/em\u003emutations were present in at least some tumors, we conducted \u003cem\u003eBRAF\u003c/em\u003e genotyping on each tumor. Pretreatment tumor biopsy samples were digested to isolate DNA, and \u003cem\u003eBRAF\u003c/em\u003e was amplified via PCR. Sanger sequencing was used to determine the \u003cem\u003eBRAF\u0026nbsp;\u003c/em\u003esequence. The tumor samples were heterogeneous, but any detectable level of the target gene mutation (thymine to adenine at the indicated position, \u003cstrong\u003eFigure 7B\u003c/strong\u003e) was considered sufficient to classify the tumor as \u003cem\u003eBRAF\u003c/em\u003e mutant positive. Comparison of the tumor volume changes demonstrated a statistically significant difference (p = 0.01) between treatment outcomes for \u003cem\u003eBRAF\u0026nbsp;\u003c/em\u003ewildtype (WT) versus mutant (p.V595E) tumors, with the \u003cem\u003eBRAF\u003c/em\u003e p.V595E expressing tumors shrinking to a greater degree, and with greater frequency, than \u003cem\u003eBRAF\u003c/em\u003e WT tumors \u0026nbsp;(\u003cstrong\u003eFigure 7C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study demonstrates that trametinib, given orally at home daily, can have a substantial effect in reducing the size of ~\u0026thinsp;30% of OSCC tumors in dogs. Only a single case so far resulted in a complete response, with no tumor remaining visible by CT. Two tumors did have nearly complete responses however, with \u0026gt;\u0026thinsp;90% reduction in tumor volume, and it is possible that a complete response might have occurred following a longer treatment duration. Thus, we currently consider trametinib treatment to be a neoadjuvant intervention, which might help to reduce the size of some tumors, allowing them to be better surgical candidates. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, patient sex, tumor location, patient age, and initial tumor size all failed to predict drug efficacy. However, \u003cem\u003eBRAF\u003c/em\u003e mutational status was predictive, with canines having \u003cem\u003eBRAF\u003c/em\u003e mutant tumors showing significantly better response to drug than canines with \u003cem\u003eBRAF\u003c/em\u003e wildtype tumors (p\u0026thinsp;=\u0026thinsp;0.01). The ORR for \u003cem\u003eBRAF\u003c/em\u003e WT tumors was only 38%, compared to 86% for \u003cem\u003eBRAF\u003c/em\u003e p.V595E tumors. Moreover, of the tumors that responded to drug, \u003cem\u003eBRAF\u003c/em\u003e WT tumors saw only an average of 7% reduction in volume (median change 3% volume increase), while \u003cem\u003eBRAF\u003c/em\u003e p.V595E tumors saw an average volume reduction of 71% (median change 89% volume reduction).\u003c/p\u003e \u003cp\u003eIn humans trametinib is typically prescribed for \u003cem\u003eBRAF\u003c/em\u003e-mutant tumors, alongside the BRAF inhibitor dabrafenib\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In dogs, we have evidence that \u003cem\u003eBRAF\u003c/em\u003e mutation may drive some OSCC tumor growth, but do not know what activating mutations or signaling pathways drive other OSCCs\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. It is unclear to us if inhibiting MEK might only be a viable strategy when \u003cem\u003eBRAF\u003c/em\u003e has been mutated, with other driving proteins somehow bypassing MEK activity, or if perhaps other driving proteins cause drug export proteins which target trametinib to be overexpressed. Screening for \u003cem\u003eBRAF\u003c/em\u003e mutation can be accomplished via comparatively simple PCR amplification and Sanger sequencing, and we hope to see the Cornell AHDC offer \u003cem\u003eBRAF\u003c/em\u003e screening as a service in the near future.\u003c/p\u003e \u003cp\u003eWhen we designed this study, we started with a \u0026lsquo;do no harm\u0026rsquo; approach. In our experience, following a patient\u0026rsquo;s first arrival to the clinic, it takes approximately two weeks before surgery (i.e., the current standard of care) can be scheduled and conducted. Our current approach for enrolled patients has been to immediately prescribe trametinib. This is typically delivered to the owners in 1\u0026ndash;3 days, leading to roughly 12 days of drug treatment before the dog\u0026rsquo;s next examination. Notably, dogs were removed from the trial immediately if progressive disease was detected following any scheduled examination. Thus, only patients showing responsive disease were kept on the drug for the full 8-week duration. Moreover, the treatment is relatively convenient, as it can be administered at home, and so does not require routine hospital visits. It is also well tolerated: while several grade 1 and grade 2 adverse events were detected in blood chemistry panels, they were inconsistent and we consider it unlikely that they were a result of trametinib treatment. The two grade 3 events (ALP levels in dogs #4 and #20) were preceded by grade 2 elevations in ALP (both dogs) and ALT (dog #20) which were present during the initial bloodwork, suggesting liver damage may have been in place before drug was given, and making it unlikely that the drug was responsible for the grade 3 events. Some grade 1 gastrointestinal distress was reported by owners but was managed with minor additional care. Combined, this suggests that trametinib is a low-risk addition to the current standard of care for canine OSCC.\u003c/p\u003e \u003cp\u003eEthical concerns prevented our ability to use an untreated control group. CT scans to accurately assess tumor volume require anesthesia, which is a procedure with some inherent risk and expense\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and we could not ethically justify subjecting dogs to full-body anesthesia and radiation exposure simply to gather numbers on exact tumor growth rates absent drug. These concerns were magnified by the comparatively small number of dogs we were able to recruit into our study, and the reality that every control patient would have removed a treated patient from our already limited pool. Thus, we cannot make any arguments regarding whether treated tumors which did grow grew slower than they might have been expected to do otherwise. However, experience has taught us that tumors do not shrink on their own, and that dogs rarely if ever show a placebo effect after drug treatment, and so we are confident in our overall results regarding tumor drug response.\u003c/p\u003e \u003cp\u003eA small number of clinical trials examining canine OSCC have been conducted in the past 25 years. One early study examined photodynamic therapy with 2-(1-hexyloxyethyl)-2-devinylpyrooheophorbide-a (HPPH). The authors reported that they examined eleven dogs, and remaining tumor was detected in 9 of the 11 dogs (82%), with two dogs having a complete response (18%). However, following surgical intervention, eight dogs (73%) had no tumor recurrence after 17 months. The authors reported that it took up to 6 weeks to heal from treatment, that the treatment could only be provided in the hospital over several days, and that tooth loss occurred at the site of treatment\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. A more recent investigation examined hyper-fractionated radiation combined with surgery, and concluded that surgery was still the gold standard treatment, but that postoperative radiation could extend survival times in cases where clean margins could not be obtained\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. A water-soluble form of paclitaxel (trade name Paccal Vet) was used in experiments treating 32 dogs with a variety of tumors, including 3 with squamous cell carcinomas\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The drug was effective in these dogs (CR or PR), but the majority of dogs in the study were reported to experience grade 1 to grade 4 adverse events which were attributed to the treatment. The drug initially had conditional approval by the FDA, but that approval has since been revoked. Finally, a series of studies has investigated the non-steroidal, anti-inflammatory compound piroxicam either alone\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, or with cisplatin\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e or carboplatin\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. When treated with piroxicam alone, three out of seventeen dogs (18%) responded to the drug, with an additional five dogs (29%) reaching a stable disease state. In combination with cisplatin, 5 of 9 dogs (56%) showed signs of remission, although all dogs had substantial but manageable toxicity, particularly renal toxicosis. When given with carboplatin, 4 of 7 dogs (57%) showed a complete response, with 2 of 7 (29%) showing a partial response. However, mild-to-moderate gastrointestinal side effects were noted, albeit none requiring hospitalization. By comparison, trametinib treatment has a PR or CR response rate of 30%, which increases to 71% when only \u003cem\u003eBRAF\u003c/em\u003e mutant-positive tumors are considered. The drug had no notable toxic effects, and the entire treatment could be completed at home. Interestingly, our results closely mirror similar results in a human clinical trial examining mandibular ameloblastoma\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, which resulted in a mix of partial and near-complete drug responses, once again showing the incredible power of dogs as translational models of disease and disease treatment.\u003c/p\u003e \u003cp\u003eWhile we are very excited about these results, there are several key limitations to this study. Many of our key conclusions are drawn from CT measurements. These are inherently limited by occasional unclean margins in some tumors, and the reality that tumor volumes identified by CT are likely a combination of tumor tissue and surrounding inflammation, both of which impact the accuracy of the measurements. Moreover, we do not yet have longitudinal data to determine tumor recurrence, and in fact many patients were treated with surgery immediately following the conclusion of drug treatment to prevent recurrence to the greatest degree possible. While we were able to treat narrower margins, this does mean that our long-term survival data will never be directly comparable to some other studies. Because the drug treatment was given at home, our data may reflect dogs whose owners did not follow the dosing regimen correctly. Similarly, we do not account for confounding elements (e.g., dogs living in a home in which tobacco is routinely smoked, or drug doses being given at different times of day) which might have affected tumor growth, drug efficacy, or overall dog health. However, this does mean our trial reflects real-world conditions better than if all animals had been hospitalized for the duration of treatment. Further, as is common in veterinary clinical trials, we were only able to enroll a comparatively small number of dogs to the study. This is particularly a concern for \u003cem\u003eBRAF\u003c/em\u003e-based stratification. While there was a statistically significant difference in outcomes between patient populations, we would feel much more confident in this as a single-factor determinant of efficacy if we had been able to study a greater number of dogs with \u003cem\u003eBRAF\u003c/em\u003e-mutant tumors. We are seeking to address this shortcoming in the near future, and we do currently feel confident in this approach as a safe, affordable, and efficacious neoadjuvant therapy which can help shrink OSCC tumors in ~\u0026thinsp;33% of dogs prior to surgical intervention.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe MEK inhibitor trametinib is a safe, effective way to reduce the size of oral squamous cell carcinoma tumors in dogs. While treating dogs without consideration for their characteristics results in ~\u0026thinsp;30% of patients seeing a good drug response, it is likely that treating only dogs with \u003cem\u003eBRAF\u003c/em\u003e-mutant tumors will raise that rate of response to at least 70%. The drug is orally available, can be given to dogs by the owner at home, and results can be seen in as little as 2 weeks of treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWPK and SP designed the study and wrote the manuscript. Dental diplomates SP, NF, and ALW worked with residents EC, MED, and CW to conduct most patient interactions. Oncologists CEB, KRH, and SRS oversaw all drug treatment decisions. ALK acted as clinical trial coordinator. MB conducted all \u003cem\u003eBRAF\u003c/em\u003e screening, and pathologist GED conducted all histology. Radiologist ABT-D conducted and interpreted all CT scanning. WPK and PCC conducted all statistical analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all the staff at CUHA, and all the owners and dogs who participated in this study, without whom these advances would not have been possible. This material is based upon work supported by the Cornell Richard P. Riney Canine Health Center Research Grants Program, a grant made available to the College of Veterinary Medicine, Cornell University.\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest pertaining to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were reviewed and approved by Cornell University\u0026rsquo;s Institutional Animal Care and Use Committee (protocol #2023-0034). 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A. \u003cem\u003eet al.\u003c/em\u003e Evaluation of cisplatin combined with piroxicam for the treatment of oral malignant melanoma and oral squamous cell carcinoma in dogs. \u003cem\u003eJournal of the American Veterinary Medical Association\u003c/em\u003e 224, 388\u0026ndash;394 (2004). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2460/javma.2004.224.388\u003c/span\u003e\u003cspan address=\"10.2460/javma.2004.224.388\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Vos, J. P. \u003cem\u003eet al.\u003c/em\u003e Piroxicam and carboplatin as a combination treatment of canine oral non-tonsillar squamous cell carcinoma: a pilot study and a literature review of a canine model of human head and neck squamous cell carcinoma. \u003cem\u003eVeterinary and Comparative Oncology\u003c/em\u003e 3, 16\u0026ndash;24 (2005). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1476-5810.2005.00065.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1476-5810.2005.00065.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrynberg, S. \u003cem\u003eet al.\u003c/em\u003e Neoadjuvant BRAF-targeted therapy for ameloblastoma of the mandible: an organ preservation approach. \u003cem\u003eJNCI: Journal of the National Cancer Institute\u003c/em\u003e, djad232 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jnci/djad232\u003c/span\u003e\u003cspan address=\"10.1093/jnci/djad232\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"veterinary-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Veterinary Oncology](https://veterinaryoncology.biomedcentral.com/)","snPcode":"44356","submissionUrl":"https://submission.springernature.com/new-submission/44356/3","title":"Veterinary Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cancer, oral squamous cell carcinoma, canine, dog, trametinib, RAS signaling, clinical trial","lastPublishedDoi":"10.21203/rs.3.rs-6821917/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6821917/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eOral squamous cell carcinoma (OSCC) is a common and deadly disease in dogs. The current standard of care involves wide-margin surgical excision of tumor tissue, which is frequently disfiguring and can be debilitating, leading to a reduced quality of life. Recent studies have demonstrated that OSCC in dogs typically shows highly elevated RAS signaling compared to healthy gingival tissue. Here, we demonstrate that the FDA-approved drug trametinib, which is prescribed in humans for \u003cem\u003eBRAF\u003c/em\u003e-mutant melanomas, is effective in treating OSCC in dogs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Domestic companion dogs (N = 20) with spontaneously occurring OSCC tumors were recruited over a two-year period for an interventional study without concurrent controls. Dogs were prescribed 0.015 to 0.035 mg/kg trametinib daily to be given orally, and owners monitored their dog’s health at home. Treatment was continued for 8 weeks, with examinations every 2 weeks. Tumor volume was assessed by caliper measurement and by computed tomography (CT) imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Five dogs achieved a partial response (PR), and one dog had a complete response (CR) based on CT imaging at the end of 8 weeks of treatment, however cancer cells were detected by histological examination. Four dogs had stable disease (SD), and ten dogs were removed from the trial after demonstrating progressive disease (PD). Response categorization was based on R.E.C.I.S.T. criteria, and PD was typically detected by caliper measurements within 2-4 weeks of beginning treatment. The overall response rate was 50% (30% CR/PR, 20% SD). Adverse events were rare, low grade, and resolved with outpatient supportive care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eTrametinib effectively blocks the growth of canine OSCC in ~50% of dogs and is well tolerated with minimal side effects. Approximately one-third of the dogs treated showed at least partial tumor volume decrease. Considering all of our findings together, we determine that trametinib is a plausible neoadjuvant step for OSCC which shows clinical benefit for approximately half of dogs prior to surgical intervention. Overall, this work presents an effective, safe, and available targeted therapeutic approach for the treatment of OSCC tumors in dogs.\u003c/p\u003e","manuscriptTitle":"Clinical application of the MEK inhibitor trametinib in dogs with oral squamous cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 14:44:21","doi":"10.21203/rs.3.rs-6821917/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-18T08:09:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-18T04:48:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-17T19:06:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212418002866681268490987591192407882768","date":"2025-07-07T19:24:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300144130940006885639979759627211094051","date":"2025-07-07T18:45:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-30T14:03:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290220156991205409548100826191898999420","date":"2025-06-24T07:10:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-12T15:46:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-11T08:53:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Oncology","date":"2025-06-10T13:32:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Veterinary Oncology](https://veterinaryoncology.biomedcentral.com/)","snPcode":"44356","submissionUrl":"https://submission.springernature.com/new-submission/44356/3","title":"Veterinary Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d913d736-c5ec-4837-ae45-a87ace246728","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-09-23T08:12:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 14:44:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6821917","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6821917","identity":"rs-6821917","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}