Increased plasma DR‐70 (fibrinogen‐fibrin degradation products) concentrations as a diagnostic biomarker in dogs with neoplasms

Abstract Background Tumor biomarkers have used widely in clinical oncology in human medicine. Only a few studies have evaluated the clinical utility of tumor biomarkers for veterinary medicine. A test for fibrinogen and fibrin degradation products (DR‐70) has been proposed as an ideal biomarker for tumors in humans. The clinical value of DR‐70 for veterinary medicine however has yet to be determined. Objectives Investigate the diagnostic value of DR‐70 concentrations by comparing them between healthy dogs and dogs with tumors. Animals Two hundred sixty‐three dogs with different types of tumors were included. Sixty healthy dogs also were recruited for comparison. Methods The DR‐70 concentrations were measured in all recruited individuals by ELISA. Clinical conditions were categorized based on histopathology, cytology, ultrasound examination, radiology, clinical findings, and a combination of these tests. Results The median concentration of DR‐70 was 2.130 ± 0.868 μg/mL in dogs with tumors, which was significantly higher than in healthy dogs (1.202 ± 0.610 μg/mL; P < .0001). With a cut‐off of 1.514 μg/mL, the sensitivity and specificity of DR‐70 were 84.03% and 78.33%, respectively. The area under curve was 0.883. The DR‐70 concentration can be an effective tumor biomarker in veterinary medicine. Conclusions and Clinical Importance Increased DR‐70 concentrations were not affected by tumor type, sex, age, or body weight. However, in dogs with metastatic mast cell tumors and oral malignant melanoma, DR‐70 concentrations were significantly increased. Additional studies, including more dogs with nonneoplastic diseases, are needed to further evaluate the usefulness of DR‐70 as a tumor biomarker.


Results:
The median concentration of DR-70 was 2.130 ± 0.868 μg/mL in dogs with tumors, which was significantly higher than in healthy dogs (1.202 ± 0.610 μg/mL; P < .0001).With a cut-off of 1.514 μg/mL, the sensitivity and specificity of DR-70 were 84.03% and 78.33%, respectively.The area under curve was 0.883.The DR-70 concentration can be an effective tumor biomarker in veterinary medicine.
Conclusions and Clinical Importance: Increased DR-70 concentrations were not affected by tumor type, sex, age, or body weight.However, in dogs with metastatic mast cell tumors and oral malignant melanoma, DR-70 concentrations were significantly increased.Additional studies, including more dogs with nonneoplastic diseases, are needed to further evaluate the usefulness of DR-70 as a tumor biomarker.

| INTRODUCTION
Neoplastic diseases affect a substantial proportion of dogs, with >50% of dogs >10 years of age developing tumors. 1 However, tumors in dogs often are not identified until more advanced stages, 2,3 complicating medical intervention for affected animals. 4,5Early detection of tumors is crucial, and biomarkers have emerged as valuable indicators for tumor detection and monitoring of tumor progression. 6,7Although early tumor diagnosis using biomarkers has gained attention in veterinary medicine, 8 the translation of biomarkers into successful clinical use has been limited. 9In previous studies in humans, fibrinogen-fibrin degradation products (FDPs), specifically DR-70, have been strongly associated with gastric and colorectal tumorigenesis, making them a potential method for early tumor detection. 10,11e clinical relevance of hemostatic abnormalities in tumorigenesis and the malignant process remains controversial.Coagulation and fibrinolysis activation within tumors have been proposed as potential mechanisms that promote tumor growth and metastasis. 12Another possible mechanism involves tumor cells releasing plasmin to facilitate invasion and metastasis by promoting fibrinolysis.The cleavage of fibrinogen leads to the release of DR-70 fragments. 11These processes suggest that tumors are a source of DR-70, and higher DR-70 concentrations positively correlate with tumor development.

Fibrinogen degradation products and fibrin degradation products
(1 of which is D-dimer) must be considered differently.Fibrinogen degradation products (fragments X, D, Y, and E) are produced from fibrinogen, whereas fibrinolysis creates more complex fragments called X-oligomers, which contain the D and E fragments in various sequences. 13,14Only fibrin polymers, mediated by factor XIII crosslinking, will produce fragments containing covalent bonds between 2 D domains (D-dimer).Therefore, D-dimer is a specific fibrin degradation product that results from fibrinolysis.Affected patients generate fibrinogen and fibrin degradation products to restrict cancer cells, and the DR-70 assay measures both fibrinogen and fibrin degradation. 10,15,16The commercial D-dimer immunoassay specifically determines the epitope on the degradation products of factor XIII-cross-linked fibrin, but fails to detect fibrinogen degradation products. 13,14As an ideal tumor biomarker, tests that only measure D-dimer will miss up to half of the actual FDPs produced by tumors. 17erefore, determination of DR-70 concentrations has gained more attention in human medical oncology.
The DR-70 concentrations have been extensively studied in human clinical medicine as a diagnostic tool for tumor detection in various malignancies, including lung, 18 tongue, 19 gastrointestinal tract, 20 colorectum, 11 esophagus, 21 and ovary. 22

| Sample collection and processing
Our study was approved by the Institutional Animal Care and Use Committee of National Taiwan University, Taipei, Taiwan (protocol code: IACUC No. NTU110-EL-00096).All samples were collected with informed client consent, and all experiments followed relevant guidelines.For the healthy controls (defined as controls), canine plasma samples from active patients without neoplastic disease or healthy dogs at the National Taiwan University Veterinary Hospital and Veterinary Medical Center, the University of Tokyo, were collected with owner consent.Dogs were designated as controls based on results from a client questionnaire and physical examination by attending veterinarians.For tumor-bearing dogs (defined as patients), individuals of any age, sex, or breed were enrolled in this study.However, patients with concurrent diseases unrelated to neoplasia and deemed to be influencing the diagnosis were not eligible.Tumor types were diagnosed by histopathology, cytology or both for all patients.For all participants, regular physical examinations, evaluation of superficial lymph nodes, and blood evaluation (CBC and serum biochemistry) were evaluated.No dogs received non-steroidal antiinflammatory drugs (NSAIDs) or corticosteroids for 14 days before blood collection.Participants previously receiving chemotherapy (patients), anti-coagulant drugs, or blood transfusion also were excluded.All dogs were fasted for at least 8 hours before blood collection, and a minimum of 0.5 mL of plasma was collected from each dog.Samples were drawn from a cephalic or jugular vein into an EDTA tube (BD Vacutainer, Franklin Lakes, New Jersey) and immediately centrifugated at 450 g for 10 minutes at 4 C. Next, supernatants were collected and centrifugated at 16 000 g for 10 minutes at 4 C.After centrifugation plasma samples were collected, transferred into new tubes, and frozen at À80 C until analysis.

| Patient selection and evaluation
We collected clinical data, including breed, age, reproductive status, and tumor diagnosis.The recruited dogs were further allocated into different types of tumors according to the histopathological or cytological analysis.Of these patient dogs, cases with ≥2 types of tumors and medical records with limited information were categorized as the "other" group.For dogs with lymphoma, the immunophenotype was determined by flow cytometry or immunohistochemistry. 23 Mast cell tumors (MCTs) were categorized by recorded grade according to the clinical staging system proposed by the World Health Organization (WHO). 24Dogs with regional or systemic metastasis were classified as stage II or above.

| Measurement of FDP using DR-70 ELISA kit
According to the manufacturer's instructions, plasma DR-70 concentrations were measured using a commercial ELISA kit (DR-70 ELISA, Uni-pharma Inc., Taipei, Taiwan). 10,16The retail kit contained a microwell plate of 96-well strips coated with affinity-purified rabbit anti-DR-70 polyclonal antibodies, a horseradish peroxidase (HRP)-labeled detection antibody, tetramethyl benzidine (TMB) substrates, diluent solution, stop solution, wash buffer, low limits of quantification control, high limits of quantification control, and 5 calibrators.Briefly, the plasma samples were diluted 250-fold using the diluent solution.Next, the diluted samples were added to the antibody-coated wells in a volume of 100 μL in duplicate.After an incubation period of 30 minutes at room temperature, the microwells were washed 5 times with 1Â wash buffer.The washed microwells then were tapped on clean absorbent paper.Next, the HRP-conjugated antibodies were added into each well in a volume of 100 μL and incubated for another 30 minutes at room temperature.After incubation, the microwells were washed 5 times with 1Â wash buffer.Next, TMB substrate solution was added to each well and incubated in the dark for 15 minutes.Lastly, the reactions were stopped by adding 100 μL stop solution to each well.The end-point of the test was read by measuring the absorbance at 450 nm using a microplate reader (SpectraMax M5 Microplate Reader, San Jose, CA, USA).A standard curve was generated from the absorbances of the 5 calibrators provided in the commercial kit.The DR-70 concentrations of plasma were quantified by interpolation from the standard curve.

| Preparation and determination of canine fibrinogen degradation products
Canine fibrinogen degradation products (cFDPs) were prepared according to a previous study. 25Briefly, 150-200 mg of canine fibrinogen (Abcam, catalog no.: ab93006, Taipei, Taiwan) was dissolved in 10 mL of 20 mmol/L triethanolamine buffer.Degradation of fibrinogen was induced by incubating the mixture in a water bath at 37 C for 2 hours, with the addition of 10 casein units of plasmin per 100 mg of fibrinogen.The fibrinogenolysis process was halted by adding 2000 kilo international units (KIU) of aprotinin per milliliter.The concentration of cFDPs was quantified by bicinchoninic acid (BCA) protein assay (Bio-Rad Laboratories, Hercules, California).As shown in Figure S1, a standard curve was generated from the absorbances of the cFDPs (red line), which shared high similarity with the 5 calibrators provided in the commercial kit (blue line).These results indicate that the commercial kit also can specifically recognize canine cFDPs.

| Statistical analysis
GraphPad Prism Software 9.0 (GraphPad Software, INC, La Jolla, California, USA) was used for all statistical analyses.Data were tested for normal distribution using the D'Agostino & Pearson test.
Because results were not normally distributed, nonparametric tests were conducted for data comparisons.The Mann-Whitney U test was used for data sets containing only 2 populations, such as controls compared with patients.The Kruskal-Wallis test for repeated measures with Dunn's multiple comparisons test was used for data sets where various groups were analyzed.To evaluate the diagnostic performance of the DR-70 assay, receiver-operator characteristics (ROC) curves were generated, along with the area under the ROC curve (AUC).The sensitivities and specificities of the DR-70 results were calculated at various threshold concentrations.Youden's index (J), a measurement for evaluating biomarker effectiveness, 26 was used to determine the cut-off for calculating sensitivity and specificity.The optimal cut-off is defined as the maximum value of J. 27,28 Data are presented as the median and interquartile range (IQR, range from the 25th to the 75th percentile).Statistical difference was defined as a P-value <.05.
Tumor types of individual dogs were diagnosed by histopathology or cytology.Of the 263 patients in the study, there were 116 lymphoma cases, 17 MCT cases, 12 melanoma cases, and several additional tumor types.Among patients, age was unknown for 46 dogs, with 217 ranging from 1 to 17 years of age (median, 9 years; mean, 8.9 years).The diagnosis of tumor types and detailed characteristics of all patients are summarized in Table S2.

| Significantly increased DR-70 concentrations in tumor-bearing dogs with high diagnostic power
The DR-70 concentrations were measured in duplicate for all recruited dogs.As shown in Figure 1A, the median DR-70 concentration in patients (2.130 ± .868μg/mL) was significantly higher than in healthy controls (1.202 ± 0.610 μg/mL; P < .0001).The ROC assessed the ability of DR-70 to distinguish clinical cases from healthy controls, and the AUC was evaluated.The AUC was 0.883 with a 95% confidence interval (CI) of 0.833 to 0.923, which indicated ideal diagnostic performance of DR-70 to discriminate between dogs with tumors and healthy controls (Figure 1B; P < .0001).

| High sensitivity and specificity of DR-70 for tumor diagnosis at an optimal concentration of 1.514 μg/mL
Additional diagnostic test variables were calculated for DR-70.The diagnostic sensitivity and specificity of the DR-70 varied by the selected cut-off.According to the results, with cut-offs extending from 1.494 to 1.536 μg/mL, sensitivity was between 82.13% and 84.79%, whereas specificity was between 73.33% and 78.33% (Table 3).Based on the maximum value of J, an optimal cut-off of 1.514 μg/mL with a sensitivity of 84.03% (95% CI, 79.03%-88.24%)and specificity of 78.33% (95% CI, 65.80%-87.93%)was selected.
These results indicate that at a concentration of 1.514 μg/mL, DR-70 has a high potential to be designated as a biomarker for distinguishing dogs with and without tumors.

| Increased DR-70 concentrations in dogs with inflammation and acute diseases
To elucidate DR-70 concentrations in dogs without neoplasia, we enrolled 24 patients without tumors at National Taiwan University Veterinary Hospital (Table S3).The DR-70 concentrations were significantly increased in dogs with tumors (P < .0001)and in nontumor-bearing dogs (n = 24, 1.937 ± 0.424 μg/mL; P < .001)compared to controls.No difference was found between dogs with tumors and non-tumor-bearing dogs (Figure 4A).We further allocated    With regard to the similarity between tumors in humans and dogs, as canine genome sequencing became available, humans and dogs were found to be more genetically similar than humans and rodents. 29Regarding cancer-related genes, several studies have shown high similarity between humans and dogs, such as mTOR, RBRCA, retinoblastoma, p16, p53, and other genes. 30,31In addition, tumors in humans and dogs have a high degree of histopathological similarity, and the pattern of distant metastasis also is similar in some tumor types.For example, melanoma is prone to lung metastasis in dogs. 32In addition, many similarities exist between human breast cancer and mammary cancer in dogs.For example, human and canine triple-negative breast tumors have more rapid recurrence times and higher recurrence rates than do endocrine receptor-positive breast tumors.There is also a higher rate of invasion of surrounding tissues and metastases.These characteristics also are found in MGTs in dogs. 33This similarity likely is because the interaction between tumor growth and the immune system is identical, and even relapse after treatment and drug resistance are similar in dogs. 34In addition, humans and dogs inhabit the same environment for long periods, making environmental carcinogens a possible commonality for tumors in both humans and dogs. 35In the past, experimental data from dogs could guide the treatment of cancers in humans and cancer research.
As cancer treatment in humans has made substantial advancements, these advances can be used to inform cancer treatment in dogs, with tumor markers used for humans also being utilized for cancer diagnosis in dogs.
Few studies have described increased DR-70 concentrations in dogs with tumors.Several studies focused on the upstream product, fibrinogen, are widely reported in veterinary medicine.One study reported abnormal coagulation parameters in dogs with tumors, such as hyperfibrinogenemia. 36Dogs with tumors often present with abnormal hemostatic parameters, including increased fibrinogen concentrations. 37However, these previous studies focused on reporting ).We used the WHO grading system to grade these samples.Thus, additional investigations are recommended to verify that similar differences are found when another grading system, such as a 2-tier histologic grading system, is applied.finding.We also found that dogs with oral malignant melanoma had increased DR-70 concentrations, significantly higher than those with cutaneous melanoma.Almost all cases of cutaneous melanomas were benign, whereas melanomas in the oral cavity were malignant in our study.This finding indicates the possibility of using DR-70 concentrations to discriminate between less aggressive melanoma and more malignant melanoma.However, the medical records did not report the malignant status of 2 cases with cutaneous melanomas.
Therefore, additional studies with detailed histopathological examinations for each melanoma case should be performed to support our findings.
Our study had some limitations.First, this preliminary investigation involved several types of tumors in dogs, and additional studies that focus on certain types of tumors are needed.Furthermore, the retrospective design of our study restricted our findings.Cohort and case-control prospective studies would be useful.Although more investigations are needed to address these issues, our study did illustrate that DR-70 has excellent potential to be considered as a tumor biomarker in veterinary medicine. In

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.

ORCID
Yu-Shan Wang https://orcid.org/0000-0002-3774-1422 Chen-Si Lin https://orcid.org/0000-0003-2254-8344 These studies indicate significant correlation between increased DR-70 concentrations and tumor formation across multiple types of tumors.However, the accuracy and utility of DR-70 as a tumor biomarker in veterinary medicine remain unknown.Additionally, no previous publication in veterinary oncology has explored DR-70 concentrations across different tumor types stratified by sex, age, and breed.Therefore, we aimed to investigate the diagnostic potential of plasma DR-70 concentrations in dogs with tumors compared to healthy individuals.Furthermore, we sought to determine DR-70 concentrations among different tumor types and assess the categorization of DR-70 concentrations based on other variables in tumor-bearing dogs.
When all cases with tumors were considered together compared to healthy controls, the DR-70 concentrations significantly increased in the patient group.Plasma DR-70 concentrations were evaluated for tumors with at least 5 cases each.We then analyzed the DR-70 F I G U R E 1 Increased DR-70 concentrations in the plasma of dogs with tumors.(A) The median concentrations of DR-70 in healthy controls and dogs with neoplasms were 1.202 ± 0.610 and 2.130 ± 0.868 μg/mL, respectively.(B) ROC curve demonstrates the area under the ROC curve (AUC) for all dogs with tumors compared to healthy animals.The AUC is 0.883 under a 95% confidence interval of 0.842-0.923,with a significant difference (P < .0001).Data were described as median ± interquartile range and analyzed by the Mann-Whitney U test.P values <.05 were considered significant.****P < .0001.concentrations of dogs with the most common types of tumors, including lymphoma (n = 116), MCT (n = 17), melanoma (n = 12), transitional cell carcinoma (TCC, n = 8), and mammary gland tumor (MGT, n = 5).The DR-70 concentrations of lymphoma, MCT, melanoma, TCC, and MGT were 2.135 ± 0.820, 1.873 ± 0.660, 1.777 We next evaluated whether increased DR-70 concentrations were present for B-cell (BCL) and T-cell (TCL) lymphoma.Immunophenotyping information was available for 95 cases, and DR-70 concentrations were compared between 2 immunophenotype groups and healthy control dogs.The DR-70 concentrations were significantly increased in dogs with BCL and TCL compared to healthy controls.The median DR-70 concentrations for BCL and TCL were 2.223 ± 0.713 μg/mL (n = 76) and 1.891 ± 0.770 μg/mL (n = 19), respectively.However, no significant difference was found in these F I G U R E 2 DR-70 concentrations across multiple tumor types relative to the healthy cases.(A) DR-70 concentrations were categorized by various common tumor types, such as lymphoma (n = 116), mast cell tumor (n = 17), melanoma (n = 12), TCC (n = 8), and mammary gland tumor (n = 5).(B) DR-70 concentrations for lymphoma cases by immunophenotyping (B-cell or T-cell lymphoma).(C) DR-70 concentrations for dogs with mast cell tumors according to grades (T0-T1 or T2-T4).(D) DR-70 concentrations for melanoma cases by location (cutaneous or oral region).Data were described as median ± interquartile range and analyzed by the Mann-Whitney U or Kruskal-Wallis test.P values <.05 were considered to indicate statistical significance.*P < .05;**P < .01;***P < .001;****P < .0001;n.s., no significant difference.2populations (P > .05;Figure2B).The MCTs were divided by recorded grade, and this information was available for 17 dogs with MCTs.Dogs with grade 0 and 1 MCTs (n = 9, 1.689 ± 0.586 μg/mL) had lower DR-70 concentrations compared to those with grades 2, 3, and 4 (n = 8, 2.275 ± 0.680 μg/mL; P < .01;Figure2C).Among melanoma cases, 2 main groups were recorded: cutaneous (n = 6) and oral (n = 6) malignant melanomas.The cutaneous melanoma group had 4 benign cases; the other 2 were not characterized.All of the oral melanoma cases were diagnosed as malignant tumors.The DR-70 concentrations were significantly lower in dogs with cutaneous melanoma (n = 6, 1.518 ± 0.297 μg/mL) compared to those with melanoma (n = 6, 2.096 ± 0.215 μg/mL; P < .01; Figure Collectively, dogs with metastatic MCTs or oral malignant melanoma had increased DR-70 concentrations.These results indicate that DR-70 concentrations are positively correlated with tumor malignancy. inflammatory diseases) according to general evaluation by clinical veterinarians (Figure4B).The DR-70 concentrations in dogs with inflammation (n = 15, 1.987 ± 0.295 μg/mL; P < .001)were significantly higher than those in the controls, but were not different compared to dogs with tumors.In dogs without inflammation (n = 9, 1.639 ± 0.970 μg/mL), DR-70 concentrations were not different concentrations in controls, dogs with inflammation, and dogs with tumors.As shown in Figure4C, the non-tumor-bearing dogs were divided into 2 populations again, and stratified by disease phase (acute or chronic).Increased DR-70 concentrations were found in dogs with acute diseases versus controls (n = 12, 2.064 ± 0.366 μg/mL; P < .0001),and not significantly different from dogs with tumors.Dogs with chronic diseases exhibited relatively low DR-70 concentrations (n = 12, 1.646 ± 0.597 μg/mL) compared to those with tumors (P < .05).Notably, no significant difference was found between dogs with acute and chronic diseases (P = .05).Increased DR-70 concentrations found in dogs with inflammatory diseases might result from an acute phase response.We speculated that DR-70 concentrations might return to normal after resolution of inflammation.

a
American Kennel Club (AKC) defined the breed weight chart and breeds.T A B L E 3 Sensitivity and specificity of DR-70 for diagnosis of tumors at different cut-off concentrations.Developing biomarkers to diagnose tumors is essential in human and veterinary medicine.We demonstrated that DR-70 concentrations are practical biomarkers for distinguishing dogs with or without tumors.In our study, DR-70 concentrations were significantly higher in the tumor-bearing dogs than in the healthy controls.Plasma DR-70 concentrations were further analyzed for certain types of tumors (n ≥ 5 for each type of tumor), but no significant differences were found among these groups.Notably, dogs with metastatic MCT or oral malignant melanoma had higher concentrations of DR-70 than did those with local MCT or cutaneous melanoma.These findings indicate that DR-70 concentration is a useful diagnostic biomarker in dogs with tumors.
abnormal coagulation tests or hemostatic alterations in dogs with neoplastic diseases, and the clinical relevance and utility of DR-70 concentrations were rarely described.To the best of our knowledge, our study is the first to report on the clinical relevance of DR-70 in veterinary oncology.F I G U R E 4 Comparisons of DR-70 concentrations in dogs with tumors and with nonneoplastic diseases.(A) Statistical analysis of DR-70 concentrations stratified by nonneoplastic diseases, (B) inflammatory status, (C) and disease progression compared with healthy and tumor dogs.Data were described as median ± interquartile range and analyzed by the Kruskal-Wallis test.P values <.05 were considered significant.n.s., no significant difference; *P < .05;***P < .001;****P < .0001.AD, acute diseases; CD, chronic diseases; HD, healthy dogs; ID, inflammatory diseases; N-ID, non-inflammatory diseases; N-TD, non-tumor dogs; TD, tumor dogs.Our study found increased DR-70 concentrations in dogs with tumors and analyzed these in certain tumors.The DR-70 concentrations of dogs with BCL and TCL were not significantly different.The underlying mechanisms of this phenomenon remain unknown.One possible explanation is the inequality of sample numbers between BCL (n = 76) and TCL (n = 19).Verifying these findings with additional cases of TCL would be useful.Standard immunophenotyping (CD3/CD5 positivity for TCL or CD19/CD21 positivity for BCL) failed to distinguish less aggressive lymphoma from more malignant tumors.For example, in dogs with TCL, some samples were low-grade (indolent), advanced TCL, or different subtypes of malignancies.However, because of the restrictions of our study's retrospective design, we could not analyze the DR-70 concentrations among different subtypes of TCL.Previous veterinary studies have shown that hemostatic parameters are higher in aggressive tumors. 38-41Hence, we speculate that DR-70 concentrations might increase more in malignant subtypes of lymphoma than in other lymphoma phenotypes.If aggressiveness was to be characterized, it is expected that DR-70 concentrations would be affected in specific populations.Lastly, although it is also possible that DR-70 concentrations in dogs with BCL and TCL are not significantly different, additional studies are needed to expand on these considerations.Our study achieved a sensitivity of 84.03% and a specificity of 78.33% in differentiating tumor-bearing dogs from healthy controls using DR-70 concentration at a cut-off of 1.514 μg/mL.Among 195 patients, 38 cases were below this cut-off, including 3 of 9 dogs with T0-T1 MCT and 3 of 6 dogs with cutaneous melanoma.Notably, all 8 dogs with T2-T4 MCT and 6 cases of oral malignant melanoma had DR-70 concentrations >1.514 μg/mL.This finding was expected because DR-70 concentrations correlate with tumor grading and malignancy.Furthermore, dogs with metastatic MCTs (T2-T4) had significantly higher plasma concentrations of DR-70 than those without metastasis (T0-T1

Furthermore, we found
that DR-70 concentrations might positively correlate with overall survival time in dogs with MCTs.Dogs with T0-T1 MCTs exhibited longer median overall survival time (n = 5; median, 569 days; range, 126-1065 days) than did those with T2-T4 MCTs (n = 5; median, 222 days; range, 108-506 days).However, we did not present data this because of missing medical records and different medical interventions in these dogs.Therefore, welldesigned studies with larger sample sizes are needed to clarify this conclusion, DR-70 concentrations were increased in the investigated patients, and dogs with metastatic MCTs or oral malignant melanoma had higher concentrations of DR-70.These results indicate that DR-70 could be a useful tumor biomarker.Additional investigations are recommended for confirmation of the accuracy and clinical relevance of DR-70 as a tumor biomarker.ACKNOWLEDGMENT This research was funded by National Taiwan University (112L891503).The authors thank Mr. Winston W.T. Han for the proof and editing of this manuscript.The data presented in this study are available on request from the corresponding author.CONFLICT OF INTEREST DECLARATION Chun-Hung Wu and Chin-Hao Hu are compensated as consultants to Uni-Pharma Co-Ltd.Uni-Pharma Co-Ltd employs Chueh-Ling Lu and Chiao-Lei Cheng.Yu-Shan Wang is the general manager at Uni-Pharma Co-Ltd and holds vested equity in Uni-Pharma Co-Ltd.Yu-Shan Wang is also an inventor of multiple patent applications covering technologies for cancer diagnostics.No other authors declare a conflict of interest.OFF-LABEL ANTIMICROBIAL DECLARATIONAuthors declare no off-label use of antimicrobials.INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATIONAll samples were collected with informed client consent.This study was approved by the IACUC of National Taiwan University, Taipei, Taiwan (protocol code: IACUC No. NTU110-EL-00096).
Plasma DR-70 concentrations in dogs with tumors are not affected by sex, age, or body weight

1
Comparisons of DR-70 concentrations by sex, age, and breed in dogs with tumors.
the non-tumor-bearing dogs into 2 groups (inflammatory or non- DR-70 concentrations are categorized by breeds of dogs with tumors. Note: Data were presented as median (interquartile range).