High Prognostic relevance of toluidine blue stain score in canine cutaneous mast cell tumor

High Prognostic relevance of toluidine blue stain score in canine cutaneous mast cell tumor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report High Prognostic relevance of toluidine blue stain score in canine cutaneous mast cell tumor Avantika Srivastav, Shyama N. Prabhu, Neeraj Kumar Gangwar, Ajay Pratap Singh, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3881523/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The aim of the study was to characterize the canine cutaneous mast cell tumor based on cytomorphology and to assess the significance of cytology, histology and immunohistochemistry in its diagnosis and prognosis. The tumors were cytologically and/or histologically graded as high or low. The tumors were also graded based on the cytoplasmic granularity by toluidine blue staining method and immunostaining pattern shown by cKIT. A comparison of various parameters like breed, age, sex, tumor location size, granulation, cytological/histological grade, cKIT staining pattern and cKIT IS between the dead and live animals was made. Based on histology/cytology, 12 tumors were of high grade and four were of low grade. Median survival time for the high grade tumor was found to be 5 months. Immunocytochemistry revealed intense cytoplasmic diffuse pattern followed by stippled and membraneous patterns. No significant difference was noted for tumor location, size, sex, histological grade, cKIT Immunostaining pattern and IS between the two prognostic groups dead and live. The KIT staining pattern was not much informative for the prognosis of canine cutaneous mast cell tumors in this study. But a significant difference between the two groups was noted for the cytoplasmic granularity demonstrated by toluidine blue stain. The toluidine blue staining score served as better prognostic indicator with poor granulation favouring a worse prognosis. Therefore, toluidine blue staining may be considered a cost effective, easier and rapid method to perform as compared to immunostaining in the prognosis of canine cutaneous mast cell tumour. Cutaneous mast cell tumors canine immunocytochemistry grading Figures Figure 1 Figure 2 Introduction Mast cell tumor (MCT) accounts for 16–21% of all skin neoplasms in dogs presented either as solitary masses or as metastatic disease (Shoop et al. 2015). It frequently affects the trunk, perineal region, extremities, head and neck (Bae et al. 2020). The biological behavior of MCT is often highly unpredictable in many clinical cases. Several prognostic markers for these tumors have been studied by earlier workers, some of which include grading, staging, cKIT expression, kit gene mutations, Ki 67 expression, nodal metastasis etc. However none of them are considered 100% accurate in predicting the outcome. MCT is histologically graded using a 3-tier (Patnaik et al. 1984) and 2-tier grading system (Kiupel et al. 2011). Grading of MCTs can also be done on the basis of cytological features (Camus et al. 2016). The immunoexpression of cKIT has been well documented in canine MCT (da Costa et al. 2015). In IHC, different patterns of cKIT (CD117) are associated with mutations in the c-kit proto-oncogene which are considered as indicators of the biological behavior of MCT (Webster et al. 2006). These tumors continue to behave aggressively and therapy is still difficult despite numerous research conducted on prognostic variables in canine MCT. Therefore the following work has been carried out considering a wide range of criteria like age, sex, tumour location, ulceration, grade, granulation, cKIT staining pattern and cKIT immunoscore and compared with the prognostic outcome in two groups of animals. Material and Methods The dogs that were presented to the Veterinary Clinical Complex, College of Veterinary Science and Animal Husbandry, Uttar Pradesh Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, (DUVASU), Mathura, India with a history of cutaneous mass or swelling were evaluated. A total of 20 biopsy samples from 20 different dogs diagnosed with cutaneous/subcutaneous mast cell tumor during the period from January 2021 to December 2022, were selected for the present study. Patient data, including breed, age, gender, lesion location, size, consistency, and ulceration, were recorded. A cytological examination was performed using a fine needle, and the smears were stained with Giemsa stain. (Cowell et al. 2007). All the experimental protocols were undertaken following approval of the Institutional Animal Ethics Committee, U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001 (IAEC/21/24 vide letter no. 133/IAEC/21 dated 18.03.2022 and IAEC/ 22/19 vide letter no. 136/IAEC/22 dated 31.01.2021). The positive slides for MCT were cytologically graded as high grade or low grade as per Camus et al. (2016). Wherever tissue biopsy samples were available (8/20), the tissue samples were fixed in 10% Neutral Buffered Formalin and routinely processed for histological analysis. Sections of 5-micron thickness were cut in a microtome and stained with hematoxylin and eosin. A two-tier grading system was used in histopathological sections, as proposed by Kiupel et al. (2011). Toluidine blue stain Cytological smears and histopathological tissue sections were stained with the toluidine blue special stain to differentiate mast cells (Sridharan and Shankar, 2012). Slides were hydrated in alcohol and distilled water for 3 minutes, then subjected to toluidine blue working solution (pH 2.0 ~ 2.5) for 2–3 min. Sections were washed in distilled water, dehydrated in alcohol, cleared in xylene, and mounted with cover slips. The granularity in mast cells was graded based on the criteria as per Sabattini and Bettini (2010). Score 1 (low density of granules in most neoplastic cells); score 2 (moderate density in most cells or high density in less than half the cells) or score 3 (high density in most cells). Immunocytochemistry/ Immunohistochemistry For immunocytochemistry (ICC), fine needle aspiration of suspected samples was carried on the 3-aminopropyltriethoxysilane (APES) coated slides that were then fixed in chilled acetone for 5 minutes. For IHC, the tissue sections of 5-micron thickness were taken on APES coated slides. Heat induced epitope retrieval (HIER) was done with 10 mM citrate buffer (pH 6.0) in a microwave (600 W for 25 min) followed by treatment with 3% hydrogen peroxide (H 2 O 2 ) for 10 min. Blocking was done with 2% BSA for 10 min. The slides were then incubated with rabbit polyclonal anti-canine c-kit antibodies with 1:20 dilution and incubated overnight at 4 0 C. The slides were then washed with PBS and incubated with HRPO conjugated anti-mouse secondary antibody and anti-rabbit IgG secondary antibody @ 1:50 dilution at room temperature for 2 hrs. DAB chromogen substrate was added to detect the immunoreactivity. The KIT pattern was assessed in immunostained sections as diffuse, focal or stippled and membraneous. The immunoscoring (KIT IS) was also done as per the criteria described by Sabattini and Bettini (2010) as the product percentage of positive cells (1, ≤ 10%; 2, 11 to 30%; 3, 31 to 60% or 4, > 60%) and intensity of labelling (1, low; 2, moderate and 3, high). Survival data: The status of the patients after diagnosis/ treatment was recorded throughout the period of study via telephonic interview with the owners. The date of diagnosis was used to calculate survival time, with 31 December 2022 as the data censor date. Dogs that died before that date were recorded as events, while those alive or failed to return or follow up before that date were censored. A Kaplan Meier survival plot was generated using Graph pad prism 8.0 software. Statistical analysis: The animals were divided into two groups based on clinical outcome that is dead and alive. Various parameters like breed, age, sex, tumour location, size, multiplicity, ulceration, consistency and cytology/histology grade were tested for significance using chi-square, Fisher exact test and logistic regression. For the cytoplasmic granulation, ICC/IHC cKIT pattern and cKIT IS, between the two groups of prognosis, the Mann Whitney U test was employed. All tests had significance set at P < 0.05, and SPSS 27.0 software was used for statistical analysis. Results Signalment MCT was found in both male and female dogs, with common breeds including Labrador retriever, Mongrel, German shepherd, Beagle, Pug, Pomeranian, Pit bull, and Great Dane. The average age of MCT in dogs was 8 years, ranging from 4 to 13 years. MCTs were found to be more common in hind limbs (9/20), followed by trunk, neck, inguinal, and scrotal areas. Two cases showed multiple site involvement. Size of the mast cell tumors recorded, ranged from 2.3 cm to 10 cm (in case of the tumors involving multiple sites, the area having tumor of largest diamensions were chosen for grading). Most of the tumours (93.33%) were over 3 cm in size and 73.33% were non-ulcerated. Cytology and histopathology The cytoplasmic granulation seen was either dense (Fig. 1a), moderate (Fig. 1b), or poor (Fig. 1c). The cytological grading showed that 16/20 were high-grade tumors with poor, moderate or dense granulation and nuclear details like mitotic figures, nuclear pleomorphism, binucleation, and more than 50% anisokaryosis. The low-grade tumors (4/20) showed dense granulation without these features. Histopathology revealed a low-grade tumor in one case (1/8) with densely granulated mast cells in subepidermal region, while the rest (7/8) were high-grade with round to pleomorphic tumorous cells. Toluidine blue staining revealed that 6/20 cases were poorly granulated with a score 1 (Fig. 1d); 9/20 had a score 2 (Fig. 1e) and 5/20 had a score 3 (Fig. 1f). Immunocytochemistry/ Immunohistochemistry The samples were positive for c Kit immunostaining showing three patterns namely diffuse cytoplasmic (Fig. 1g) in most of the cases (12/20); focal/stippled (7/20) (Fig. 1h) and membraneous (1/20) (Fig. 1i) patterns. Survival curve: A total of 20 dogs met the inclusion criteria. There were 8 deaths in total related to mast cell tumor and 12 patients were alive at the end of the study. Out of the 20 cases 19 (80%) were graded as high grade MCT and only 4 (20%) were graded as low grade. Figure 2 shows the Kaplan mayer plot for the mortality due to mast cell tumors in 20 dogs. The median survival time for LG tumors was undefined, while for HG tumors, it was estimated to be 5 months. Table 1 . Shows the comparison of various parameters between two groups alive and dead and their significance. The mean age of occurrence was 8 years in both groups, and there was no significant relationship between age and prognosis (P = 0.42). Tumor location had no relation with prognosis (P = 0.337). So also tumor size, multiplicity and presence or absence of ulceration had no prognostic relevance in this study with p values 0.394, 0.76 and 0.648 respectively. But logistic regression analysis between the two prognostic groups, taking sex and tumor location into consideration, showed that the odds ratio was more for males (OR = 1.7) and location of tumor on the trunk region (OR = 1.3). Nevertheless to establish this fact studies involving a larger number of patients have to be done. The distribution of granules or granulation in tumor cells demonstrated by toluidine blue stain score showed a significant difference (0.035) between the two prognostic groups. The less differentiated the tumor cells (poor granulation), the poorer the prognosis. However no significant difference was observed when cytology/histology grade, cKIT pattern and KIT IS were considered (p = 0.068, p = 0.418 and p = 0.077 respectively). Table 1 A comparison of various clinicopathological parameters with the outcome in twenty dogs diagnosed with cutaneous mast cell tumours. Parameters Alive (24 months post diagnosis) Dead within the 24 months study period P value Number 12 8 Gender (male: female) 6:6 4:4 1.0 Age years: mean (range) 8 (6–11) 8 (4–13) 0.42 Location Limbs Trunk Others (head, neck etc.) 7 4 1 2 5 1 0.337 Size (cm) 3–5 > 5–7 > 7 2 8 1 1 1 4 3 0 0.394 Multiplicity Solitary Multiple locations 11 1 7 1 0.76 Ulceration Non-ulcerated Ulcerated 10 2 6 2 0.648 Toluidine blue stain score Well granulated Moderate Poor 4 7 1 1 2 5 0.035 Histology/cytology Grade HG LG 8 4 8 0 0.068 cKIT pattern stippled diffuse membraneous 4 8 0 3 4 1 0.418 cKIT IS mean (range) 7.33(3–12) 8.75 (6–12) 0.077 Discussion Previous studies have reported equal prevalence of MCT in both sexes, with an increased incidence in Labrador retrievers (Patnaik et al. 1984; Camus et al. 2016). MCT are most commonly seen in the animals of the age group 6–11 years (Villamil et al. 2011). The most common sites of MCT were hindlimbs and trunk regions, with other locations including inguinal regions, head, neck, and abdomen as reported by earlier workers (Bae et al. 2020). Previous studies have associated poorer prognosis with ulceration, erythema, pruritus, and larger tumor size > 3cm diameter (Hahn et al 2004; Mullins et al. 2006). However this study showed no association of tumor ulceration or size with prognosis. Grading of the tumor was done using both cytology and/or histopathology because cytological characteristics of MCTs are more conclusive than the histological pattern. The poorly granulated anaplastic cells are more easily visible in cytology because of the greater resolution of cytoplasmic granules (Kiupel and Camus, 2019). Histological grading is regarded as an approved prognostic tool for canine cutaneous MCT (Murphy et al. 2004). Kuipel 2-tier grading is considered to be the current standard for MCT grading. The tumor grade showed no significant difference between the two prognostic groups (p = 0.068) in our study. Some studies have reported high grade solitary mast cell tumors (as per Kiupel’s criteria), to have favourable prognosis or manifest unpredictable biological behavior, regardless of grading (Moore et al. 2020). Histological grading has a limited prognostic value especially for tumours of intermediate differentiation as these tumors tend to behave erratically (Weisse et al. 2002). This could be due to the fact that there may be variation between pathologists in the assignment of various grades to the tumors, as it depends on subjective parameters like cellular or nuclear morphology, invasiveness etc. (Northrup et al. 2005). Toluidene blue is the most widely used dye to stain the mast cells (Ribatti, 2018). Demonstration of connective tissue mast cells has been done effectively using toluidine blue stain at an acidic pH (Grigorev and Korzhevskii, 2021). The cytoplasmic granulation based on toluidine blue stain score showed a significant difference between the two prognostic groups. Poor granulation or less differentiation was associated with an unfavourable prognosis. A similar result was obtained in an earlier study where loss of differentiation in neoplastic feline mast cells, accompanied by a reduced density of cytoplasmic granules, correlated with an unfavorable prognosis (Sabattini and Bettini, 2010). The c-kit immunostaining showed different types of staining patterns i.e., membrane-associated pattern, focal cytoplasmic pattern, and diffuse cytoplasmic pattern as reported by earlier studies (Sailasuta et al. 2014). The c-kit immunostaining is considered a useful factor in the prognosis of canine MCT by many earlier studies (Vascellari et al. 2013). However, the cKIT staining pattern as well as KIT IS showed no association with the clinical outcome in our study. Earlier studies have reported that KIT staining patterns and aberrant KIT expression had no prognostic relevance supporting our findings (Giantin et al. 2012; Casagrande et al. 2013). Inspite of many research studies carried out on the prognosis of canine cutaneous mast cell tumors, these tumors still continue to be challenging to get treated, mainly due to their unpredictable biological behavior. Studies involving a larger group of animals may provide better insights in establishing the prognostic efficacy of the various parameters studied like multiplicity of lesions, tumor size, age, ulceration, grade and cKIT immunostaining. Also, inorder to establish reliable and routine prognostic markers for the canine cutaneous mast cell tumors, other factors of prognostic relevance like staging, recurrence, speed of growth, cytological pleomorphism or differentiation, along with mutations in KIT gene may together be taken into consideration. Grading based on toluidine blue stain score offers promising prognostic inputs in the canine cutaneous mast cell tumors as per our study. Therefore, it can be considered as a point of care test with less turnaround time, cost effective and easier to perform as compared to the immunostaining. Declarations Funding This research was funded by the College of Veterinary Science and Animal Husbandry, DUVASU Mathura, Uttar Pradesh India. Acknowledgements We would like to express our extreme thanks for the indispensable assistance rendered by The Dean (DUVASU), Mathura for official support in providing us with all the necessary facilities during the entire period of this research. Conflict of Interest There are no conflicts of interest with respect to the research, authorship or publication of this research article. Author Contribution AS- conducted the experimentSNP; planned the design of work and manuscript writingNKG & RS Experimental work planAPS: Manuscript writingSP: surgical resection of tumoursJKC &AK: Statistical analysisKG & YB: Sample collection and experimental workSC & AS: provision of chemicals and manuscript writingAll the authors have equally contributed to this research work References Bae S, Milovancev M, Bartels C, Irvin VL, Tuohy JL, Townsend KL, Leeper H (2020) Histologically low‐grade, yet biologically high‐grade, canine cutaneous mast cell tumors: A systematic review and meta‐analysis of individual participant data. Vet comp oncol 8: 580-589. Camus MS, Priest HL, Koehler JW, Driskell EA, Rakich PM, Ilha MR, Krimer PM (2016). Cytologic criteria for mast cell tumor grading in dogs with evaluation of clinical outcome. Vet pathol 53:1117-1123. Casagrande TAC, Barros LMDO, Fukumasu H, Cogliati B, Chaible LM, Dagli MLZ, Matera JM (2013). The value of molecular expression of KIT and KIT ligand analysed using real-time polymerase chain reaction and immunohistochemistry as a prognostic indicator for canine cutaneous mast cell tumors. Vet Comp Oncol 13: 1–10. Cowell RL, Tyler RD, Meinkoth JH, DeNicola DB. (2007). Diagnostic cytology and hematology of the dog and cat-E-book. Elsevier Health Sciences, Oklahoma. Giantin M, Vascellari M, Morello EM, et al. (2012). c-KIT messenger RNA and protein expression and mutations in canine cutaneous mast cell tumors: correlations with post-surgical prognosis. J Vet Diagn Invest 24(1):116-126. doi:10.1177/1040638711425945 da Costa, R. M. G. (2015). C-kit as a prognostic and therapeutic marker in canine cutaneous mast cell tumours: From laboratory to clinic. The Veterinary Journal, 205(1), 5-10. Grigorev IP, Korzhevskii DE. (2021). Modern Imaging Technologies of Mast Cells for Biology and Medicine (Review). Sovrem Tekhnologii Med.; 13(4):93-107. doi: 10.17691/stm2021.13.4.10. Epub 2021 Aug 28. PMID: 34603768; PMCID: PMC8482833. Hahn KA, King GK, Carreras JK. (2004). Efficacy of radiation therapy for incompletely resected grade-III mast cell tumors in dogs: 31 cases (1987–1998). J. Am. Vet. Med. Assoc. 224: 79–82. Kiupel M, Camus M. (2019). Diagnosis and prognosis of canine cutaneous mast cell tumors. Vet Clinics: Small Anim Pract 49: 819-836. Kiupel M, Webster JD, Bailey KL, et al. (2011). Proposal of a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior. Vet Pathol 48: 147-155. Moore AS, Frimberger AE, Taylor D, Sullivan N (2020). Retrospective outcome evaluation for dogs with surgically excised, solitary Kiupel high-grade, cutaneous mast cell tumors. Vet Comp Oncol 18: 402–408. Mullins MN, Dernell WS, Withrow SJ, Ehrhart EJ, Thamm DH, Lana SE (2006). Evaluation of prognostic factors associated with outcome in dogs with multiple cutaneous mast cell tumors treated with surgery with and without adjuvant treatment: 54 cases (1998–2004). J Am Vet Med Assoc 228: 91-95. Murphy S, Sparkes AH, Brearley MJ, Smith KC, Blunden AS. (2004). Relationships between the histological grade of cutaneous mast cell tumors in dogs, their survival and the efficacy of surgical resection. Vet Rec 154: 743-746. Northrup NC, Howerth EW, Harmon BG et al. (2005). Variation among pathologists in the histologic grading of canine cutaneous mast cell tumors with uniform use of a single grading reference. J of Vet Diagn Invest 17: 561– Patnaik AK, Ehler WJ, MacEwen EG. (1984). Canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs. Vet Pathol 21(5):469-74. doi: 10.1177/030098588402100503. PMID: 6435301. Ribatti D (2018). The staining of mast cells: a historical overview. Int Arch Allergy Appl Immunol 176(1): 55-60. Sabattini S & Bettini G (2010). Prognostic value of histologic and immunohistochemical features in feline cutaneous mast cell tumors. Vet Pathol 47(4): 643-653. Sailasuta A, Ketpun D, Piyaviriyakul P, Theerawatanasirikul S, Theewasutrakul P, Rungsipipat A. (2014). The relevance of cd117-immunocytochemistry staining patterns to mutational exon-11 in c-kit detected by pcr from fine-needle aspirated canine mast cell tumor cells. Vet Med Int 1-8. Shoop SJ, Marlow S, Church DB, English K, McGreevy PD, Stell AJ, Thomson PC, O’Neill DG, Brodbelt DC. (2015). Prevalence and risk factors for mast cell tumors in dogs in England. Canine Genet epidemiol 2: 1-10. Sridharan G, Shankar AA. Toluidine blue: A review of its chemistry and clinical utility (2012). J Oral Maxillofac Pathol. 16(2):251-5. doi: 10.4103/0973-029X.99081. PMID: 22923899; PMCID: PMC3424943. Vascellari M, Giantin M, Capello K, Carminato A, Morello EM, Vercelli A, Granato A, Buracco P, Dacasto M, Mutinelli F. (2013). Expression of Ki67, BCL-2, and COX-2 in canine cutaneous mast cell tumors: association with grading and prognosis. Vet pathol 50: 110-121. Villamil JA, Henry CJ, Bryan JN, Ellersieck M, Schultz L, Tyler JW, Hahn AW. (2011). Identification of the most common cutaneous neoplasms in dogs and evaluation of breed and age distributions for selected neoplasms. J Am Vet Med Assoc 239: 960-965. Webster JD, Yuzbasiyan-Gurkan V, Kaneene JB, Miller R, Resau JH, Kiupel M. (2006). The role of c-KIT in tumorigenesis: evaluation in canine cutaneous mast cell tumors. Neoplasia 8(2):104-11. doi: 10.1593/neo.05622. PMID: 16611403; PMCID: PMC1578516. Weisse C, Shofer FS, Sorenmo K. (2002). Recurrence rates and sites for grade II canine cutaneous mast cell tumors following complete surgical excision. J Am Anim Hosp Assoc 38: 71–3. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-3881523","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":268632656,"identity":"7796649d-b122-422e-bbd3-511f93973fe9","order_by":0,"name":"Avantika Srivastav","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Avantika","middleName":"","lastName":"Srivastav","suffix":""},{"id":268632657,"identity":"d51007d4-4d12-4e2b-ae3c-a5aa824e00e7","order_by":1,"name":"Shyama N. Prabhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYBACCQbGBiB1AMRmfABlAIEBcVqYDYjUAgZglWwSCC14gOTsw80fGP7cSeyfdvhZNU/NHTl+BuaHDxgK7uDUIs2X2CbB2PYsccbtNLPbPMeeGUs2sBkbMBg8w6lFjoexDeidw4kNtxOAWtgOJ244wAN0ocFhfFpADjucOP92+rdinn9EaJHmYWyQYAAZfjvHjJm3jQgtkj2MIL8cNt54O6dYcm7fYWPJZqBfEvBokTjD/hjkMNl5t9M3fnjz7bAcP3vzwwcf/uDWAgLMfxgYHBuADCYeMBeIE/BqgAB7EMH4gwiVo2AUjIJRMPIAAA+tWGGxPC+BAAAAAElFTkSuQmCC","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":true,"prefix":"","firstName":"Shyama","middleName":"N.","lastName":"Prabhu","suffix":""},{"id":268632658,"identity":"a21579f0-0934-4f5d-b1a5-a8220f23f414","order_by":2,"name":"Neeraj Kumar Gangwar","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Neeraj","middleName":"Kumar","lastName":"Gangwar","suffix":""},{"id":268632659,"identity":"3ce08cd6-80e7-45ff-bd73-d7bda0cd286d","order_by":3,"name":"Ajay Pratap Singh","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Ajay","middleName":"Pratap","lastName":"Singh","suffix":""},{"id":268632660,"identity":"22acaad9-646d-4557-b973-c3e282241b99","order_by":4,"name":"Renu Singh","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Renu","middleName":"","lastName":"Singh","suffix":""},{"id":268632661,"identity":"3e64e599-0ede-4f60-89d5-f1dc36adb107","order_by":5,"name":"Sanjay Purohit","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Sanjay","middleName":"","lastName":"Purohit","suffix":""},{"id":268632662,"identity":"0e8b2bf8-0295-4bc0-b265-7d3d24a36ac8","order_by":6,"name":"J. K. Chaudhary","email":"","orcid":"","institution":"Central Agricultural University, Selesih, Aizwal, 796015, Mizoram","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"K.","lastName":"Chaudhary","suffix":""},{"id":268632663,"identity":"f6f7c864-3580-4421-9d82-9c6d6be1f83a","order_by":7,"name":"Kavisha Gangwar","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Kavisha","middleName":"","lastName":"Gangwar","suffix":""},{"id":268632664,"identity":"0692b975-26bc-415b-85f8-170f029cf1d8","order_by":8,"name":"Yash Bhate","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Yash","middleName":"","lastName":"Bhate","suffix":""},{"id":268632665,"identity":"f6df87bc-7391-4562-820f-acdc068d34d0","order_by":9,"name":"Avneesh Kumar","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Avneesh","middleName":"","lastName":"Kumar","suffix":""},{"id":268632666,"identity":"f7cfe93b-681d-4eb5-912e-d5200764dd9a","order_by":10,"name":"Vinod Kumar Singh","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Vinod","middleName":"Kumar","lastName":"Singh","suffix":""},{"id":268632667,"identity":"8ca25351-da5b-4f86-a5fc-0acd163c5396","order_by":11,"name":"Soumen Choudhury","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Soumen","middleName":"","lastName":"Choudhury","suffix":""},{"id":268632668,"identity":"dec77b34-4ea3-4dd6-8cfb-7ebbbabda1e6","order_by":12,"name":"Amit Shukla","email":"","orcid":"","institution":"U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Amit","middleName":"","lastName":"Shukla","suffix":""}],"badges":[],"createdAt":"2024-01-20 12:29:16","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-3881523/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3881523/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50085897,"identity":"036b5a61-7f8a-4047-bbea-8addefd14b0e","added_by":"auto","created_at":"2024-01-24 09:35:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":264285,"visible":true,"origin":"","legend":"\u003cp\u003ea to c. Fine Needle Aspiration cytology Giemsa stain 100x. a. The tumor cells are well differentiated well granulated and uniformly round discrete cells. b. Moderately granulated pleomorphic round cells. c. Poorly granulated discrete round cells. Binucleated cells (arrow) and mitotic figures (arrow head) are also seen. d to f Toluidene blue stain 40x. d. Well differentiated densely granulated neoplastic mast cells (score=3). e Tumor with admixture of poorly granulated and densely granulated pleomorphic mast cells (score=2); f Atypical poorly granulated pleomorphic tumor cells (score=1). Cytological smear showing positive reactivity for KIT protein. g. showing diffuse and intense cytoplasmic staining pattern of tumor cells for KIT protein; h showing focal/ stippled cytoplasmic staining pattern (arrows) for KIT protein and i. shows the immunoreactivity for KIT is mainly membrane associated (arrows) ICC DAB chromogen \u0026amp; Mayer’s haematoxylin counter stain, 1000X.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881523/v1/3c9a87476a4b9ea2dc3e2b05.jpg"},{"id":50085896,"identity":"2472b744-2122-41d7-b89b-03abd5935c36","added_by":"auto","created_at":"2024-01-24 09:35:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42992,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan Meier survival plot for two histological grades of mast cell tumors (n=20). Median survival time was not reached for low grade tumors (LG) and for high grade tumors (HG) it was 5 months.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881523/v1/2060287130928244514a7159.jpg"},{"id":53286970,"identity":"016bf914-e0a5-4a4d-8061-c7c2a98795dc","added_by":"auto","created_at":"2024-03-23 00:37:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":494177,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3881523/v1/ff3ff316-b1e3-4e1b-95b3-e420329e7d12.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"High Prognostic relevance of toluidine blue stain score in canine cutaneous mast cell tumor","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMast cell tumor (MCT) accounts for 16\u0026ndash;21% of all skin neoplasms in dogs presented either as solitary masses or as metastatic disease (Shoop et al. 2015). It frequently affects the trunk, perineal region, extremities, head and neck (Bae et al. 2020). The biological behavior of MCT is often highly unpredictable in many clinical cases. Several prognostic markers for these tumors have been studied by earlier workers, some of which include grading, staging, cKIT expression, kit gene mutations, Ki 67 expression, nodal metastasis etc. However none of them are considered 100% accurate in predicting the outcome.\u003c/p\u003e \u003cp\u003eMCT is histologically graded using a 3-tier (Patnaik et al. 1984) and 2-tier grading system (Kiupel et al. 2011). Grading of MCTs can also be done on the basis of cytological features (Camus et al. 2016). The immunoexpression of cKIT has been well documented in canine MCT (da Costa et al. 2015). In IHC, different patterns of cKIT (CD117) are associated with mutations in the c-kit proto-oncogene which are considered as indicators of the biological behavior of MCT (Webster et al. 2006).\u003c/p\u003e \u003cp\u003eThese tumors continue to behave aggressively and therapy is still difficult despite numerous research conducted on prognostic variables in canine MCT. Therefore the following work has been carried out considering a wide range of criteria like age, sex, tumour location, ulceration, grade, granulation, cKIT staining pattern and cKIT immunoscore and compared with the prognostic outcome in two groups of animals.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eThe dogs that were presented to the Veterinary Clinical Complex, College of Veterinary Science and Animal Husbandry, Uttar Pradesh Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, (DUVASU), Mathura, India with a history of cutaneous mass or swelling were evaluated. A total of 20 biopsy samples from 20 different dogs diagnosed with cutaneous/subcutaneous mast cell tumor during the period from January 2021 to December 2022, were selected for the present study.\u003c/p\u003e \u003cp\u003ePatient data, including breed, age, gender, lesion location, size, consistency, and ulceration, were recorded. A cytological examination was performed using a fine needle, and the smears were stained with Giemsa stain. (Cowell et al. 2007).\u003c/p\u003e \u003cp\u003e All the experimental protocols were undertaken following approval of the Institutional Animal Ethics Committee, U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281001 (IAEC/21/24 vide letter no. 133/IAEC/21 dated 18.03.2022 and IAEC/ 22/19 vide letter no. 136/IAEC/22 dated 31.01.2021).\u003c/p\u003e \u003cp\u003eThe positive slides for MCT were cytologically graded as high grade or low grade as per Camus et al. (2016). Wherever tissue biopsy samples were available (8/20), the tissue samples were fixed in 10% Neutral Buffered Formalin and routinely processed for histological analysis. Sections of 5-micron thickness were cut in a microtome and stained with hematoxylin and eosin. A two-tier grading system was used in histopathological sections, as proposed by Kiupel et al. (2011).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eToluidine blue stain\u003c/h2\u003e \u003cp\u003eCytological smears and histopathological tissue sections were stained with the toluidine blue special stain to differentiate mast cells (Sridharan and Shankar, 2012). Slides were hydrated in alcohol and distilled water for 3 minutes, then subjected to toluidine blue working solution (pH 2.0\u0026thinsp;~\u0026thinsp;2.5) for 2\u0026ndash;3 min. Sections were washed in distilled water, dehydrated in alcohol, cleared in xylene, and mounted with cover slips. The granularity in mast cells was graded based on the criteria as per Sabattini and Bettini (2010). Score 1 (low density of granules in most neoplastic cells); score 2 (moderate density in most cells or high density in less than half the cells) or score 3 (high density in most cells).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry/ Immunohistochemistry\u003c/h2\u003e \u003cp\u003eFor immunocytochemistry (ICC), fine needle aspiration of suspected samples was carried on the 3-aminopropyltriethoxysilane (APES) coated slides that were then fixed in chilled acetone for 5 minutes. For IHC, the tissue sections of 5-micron thickness were taken on APES coated slides. Heat induced epitope retrieval (HIER) was done with 10 mM citrate buffer (pH 6.0) in a microwave (600 W for 25 min) followed by treatment with 3% hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) for 10 min. Blocking was done with 2% BSA for 10 min. The slides were then incubated with rabbit polyclonal anti-canine c-kit antibodies with 1:20 dilution and incubated overnight at 4\u003csup\u003e0\u003c/sup\u003e C. The slides were then washed with PBS and incubated with HRPO conjugated anti-mouse secondary antibody and anti-rabbit IgG secondary antibody @ 1:50 dilution at room temperature for 2 hrs. DAB chromogen substrate was added to detect the immunoreactivity. The KIT pattern was assessed in immunostained sections as diffuse, focal or stippled and membraneous. The immunoscoring (KIT IS) was also done as per the criteria described by Sabattini and Bettini (2010) as the product percentage of positive cells (1, \u0026le;\u0026thinsp;10%; 2, 11 to 30%; 3, 31 to 60% or 4, \u0026gt;\u0026thinsp;60%) and intensity of labelling (1, low; 2, moderate and 3, high).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSurvival data:\u003c/h2\u003e \u003cp\u003eThe status of the patients after diagnosis/ treatment was recorded throughout the period of study via telephonic interview with the owners. The date of diagnosis was used to calculate survival time, with 31 December 2022 as the data censor date. Dogs that died before that date were recorded as events, while those alive or failed to return or follow up before that date were censored. A Kaplan Meier survival plot was generated using Graph pad prism 8.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis:\u003c/h2\u003e \u003cp\u003eThe animals were divided into two groups based on clinical outcome that is dead and alive. Various parameters like breed, age, sex, tumour location, size, multiplicity, ulceration, consistency and cytology/histology grade were tested for significance using chi-square, Fisher exact test and logistic regression. For the cytoplasmic granulation, ICC/IHC cKIT pattern and cKIT IS, between the two groups of prognosis, the Mann Whitney U test was employed. All tests had significance set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and SPSS 27.0 software was used for statistical analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eSignalment\u003c/p\u003e \u003cp\u003eMCT was found in both male and female dogs, with common breeds including Labrador retriever, Mongrel, German shepherd, Beagle, Pug, Pomeranian, Pit bull, and Great Dane. The average age of MCT in dogs was 8 years, ranging from 4 to 13 years.\u003c/p\u003e \u003cp\u003eMCTs were found to be more common in hind limbs (9/20), followed by trunk, neck, inguinal, and scrotal areas. Two cases showed multiple site involvement. Size of the mast cell tumors recorded, ranged from 2.3 cm to 10 cm (in case of the tumors involving multiple sites, the area having tumor of largest diamensions were chosen for grading). Most of the tumours (93.33%) were over 3 cm in size and 73.33% were non-ulcerated.\u003c/p\u003e \u003cp\u003eCytology and histopathology\u003c/p\u003e \u003cp\u003eThe cytoplasmic granulation seen was either dense (Fig.\u0026nbsp;1a), moderate (Fig.\u0026nbsp;1b), or poor (Fig.\u0026nbsp;1c). The cytological grading showed that 16/20 were high-grade tumors with poor, moderate or dense granulation and nuclear details like mitotic figures, nuclear pleomorphism, binucleation, and more than 50% anisokaryosis. The low-grade tumors (4/20) showed dense granulation without these features. Histopathology revealed a low-grade tumor in one case (1/8) with densely granulated mast cells in subepidermal region, while the rest (7/8) were high-grade with round to pleomorphic tumorous cells. Toluidine blue staining revealed that 6/20 cases were poorly granulated with a score 1 (Fig.\u0026nbsp;1d); 9/20 had a score 2 (Fig.\u0026nbsp;1e) and 5/20 had a score 3 (Fig.\u0026nbsp;1f).\u003c/p\u003e \u003cp\u003eImmunocytochemistry/ Immunohistochemistry\u003c/p\u003e \u003cp\u003eThe samples were positive for c Kit immunostaining showing three patterns namely diffuse cytoplasmic (Fig.\u0026nbsp;1g) in most of the cases (12/20); focal/stippled (7/20) (Fig.\u0026nbsp;1h) and membraneous (1/20) (Fig.\u0026nbsp;1i) patterns.\u003c/p\u003e \u003cp\u003eSurvival curve:\u003c/p\u003e \u003cp\u003eA total of 20 dogs met the inclusion criteria. There were 8 deaths in total related to mast cell tumor and 12 patients were alive at the end of the study. Out of the 20 cases 19 (80%) were graded as high grade MCT and only 4 (20%) were graded as low grade. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the Kaplan mayer plot for the mortality due to mast cell tumors in 20 dogs. The median survival time for LG tumors was undefined, while for HG tumors, it was estimated to be 5 months.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Shows the comparison of various parameters between two groups alive and dead and their significance. The mean age of occurrence was 8 years in both groups, and there was no significant relationship between age and prognosis (P\u0026thinsp;=\u0026thinsp;0.42). Tumor location had no relation with prognosis (P\u0026thinsp;=\u0026thinsp;0.337). So also tumor size, multiplicity and presence or absence of ulceration had no prognostic relevance in this study with p values 0.394, 0.76 and 0.648 respectively. But logistic regression analysis between the two prognostic groups, taking sex and tumor location into consideration, showed that the odds ratio was more for males (OR\u0026thinsp;=\u0026thinsp;1.7) and location of tumor on the trunk region (OR\u0026thinsp;=\u0026thinsp;1.3). Nevertheless to establish this fact studies involving a larger number of patients have to be done. The distribution of granules or granulation in tumor cells demonstrated by toluidine blue stain score showed a significant difference (0.035) between the two prognostic groups. The less differentiated the tumor cells (poor granulation), the poorer the prognosis. However no significant difference was observed when cytology/histology grade, cKIT pattern and KIT IS were considered (p\u0026thinsp;=\u0026thinsp;0.068, p\u0026thinsp;=\u0026thinsp;0.418 and p\u0026thinsp;=\u0026thinsp;0.077 respectively).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eA comparison of various clinicopathological parameters with the outcome in twenty dogs diagnosed with cutaneous mast cell tumours.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlive (24 months post diagnosis)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDead within the 24 months study period\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender (male: female)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6:6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4:4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge years:\u003c/p\u003e \u003cp\u003emean (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (6\u0026ndash;11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (4\u0026ndash;13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003cp\u003eLimbs\u003c/p\u003e \u003cp\u003eTrunk\u003c/p\u003e \u003cp\u003eOthers (head, neck etc.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e5\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.337\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSize (cm)\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;3\u0026ndash;5\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;5\u0026ndash;7\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.394\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMultiplicity\u003c/p\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003cp\u003eMultiple locations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUlceration\u003c/p\u003e \u003cp\u003eNon-ulcerated\u003c/p\u003e \u003cp\u003eUlcerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.648\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eToluidine blue stain score\u003c/p\u003e \u003cp\u003eWell granulated\u003c/p\u003e \u003cp\u003eModerate\u003c/p\u003e \u003cp\u003ePoor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e7\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.035\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistology/cytology Grade\u003c/p\u003e \u003cp\u003eHG\u003c/p\u003e \u003cp\u003eLG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.068\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecKIT pattern\u003c/p\u003e \u003cp\u003estippled\u003c/p\u003e \u003cp\u003ediffuse\u003c/p\u003e \u003cp\u003emembraneous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.418\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecKIT IS\u003c/p\u003e \u003cp\u003emean (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.33(3\u0026ndash;12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.75 (6\u0026ndash;12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.077\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have reported equal prevalence of MCT in both sexes, with an increased incidence in Labrador retrievers (Patnaik et al. 1984; Camus et al. 2016). MCT are most commonly seen in the animals of the age group 6\u0026ndash;11 years (Villamil et al. 2011). The most common sites of MCT were hindlimbs and trunk regions, with other locations including inguinal regions, head, neck, and abdomen as reported by earlier workers (Bae et al. 2020).\u003c/p\u003e \u003cp\u003ePrevious studies have associated poorer prognosis with ulceration, erythema, pruritus, and larger tumor size\u0026thinsp;\u0026gt;\u0026thinsp;3cm diameter (Hahn et al 2004; Mullins et al. 2006). However this study showed no association of tumor ulceration or size with prognosis.\u003c/p\u003e \u003cp\u003eGrading of the tumor was done using both cytology and/or histopathology because cytological characteristics of MCTs are more conclusive than the histological pattern. The poorly granulated anaplastic cells are more easily visible in cytology because of the greater resolution of cytoplasmic granules (Kiupel and Camus, 2019). Histological grading is regarded as an approved prognostic tool for canine cutaneous MCT (Murphy et al. 2004). Kuipel 2-tier grading is considered to be the current standard for MCT grading. The tumor grade showed no significant difference between the two prognostic groups (p\u0026thinsp;=\u0026thinsp;0.068) in our study. Some studies have reported high grade solitary mast cell tumors (as per Kiupel\u0026rsquo;s criteria), to have favourable prognosis or manifest unpredictable biological behavior, regardless of grading (Moore et al. 2020). Histological grading has a limited prognostic value especially for tumours of intermediate differentiation as these tumors tend to behave erratically (Weisse et al. 2002). This could be due to the fact that there may be variation between pathologists in the assignment of various grades to the tumors, as it depends on subjective parameters like cellular or nuclear morphology, invasiveness etc. (Northrup et al. 2005).\u003c/p\u003e \u003cp\u003eToluidene blue is the most widely used dye to stain the mast cells (Ribatti, 2018). Demonstration of connective tissue mast cells has been done effectively using toluidine blue stain at an acidic pH (Grigorev and Korzhevskii, 2021). The cytoplasmic granulation based on toluidine blue stain score showed a significant difference between the two prognostic groups. Poor granulation or less differentiation was associated with an unfavourable prognosis. A similar result was obtained in an earlier study where loss of differentiation in neoplastic feline mast cells, accompanied by a reduced density of cytoplasmic granules, correlated with an unfavorable prognosis (Sabattini and Bettini, 2010).\u003c/p\u003e \u003cp\u003eThe c-kit immunostaining showed different types of staining patterns i.e., membrane-associated pattern, focal cytoplasmic pattern, and diffuse cytoplasmic pattern as reported by earlier studies (Sailasuta et al. 2014). The c-kit immunostaining is considered a useful factor in the prognosis of canine MCT by many earlier studies (Vascellari et al. 2013). However, the cKIT staining pattern as well as KIT IS showed no association with the clinical outcome in our study. Earlier studies have reported that KIT staining patterns and aberrant KIT expression had no prognostic relevance supporting our findings (Giantin et al. 2012; Casagrande et al. 2013).\u003c/p\u003e \u003cp\u003eInspite of many research studies carried out on the prognosis of canine cutaneous mast cell tumors, these tumors still continue to be challenging to get treated, mainly due to their unpredictable biological behavior. Studies involving a larger group of animals may provide better insights in establishing the prognostic efficacy of the various parameters studied like multiplicity of lesions, tumor size, age, ulceration, grade and cKIT immunostaining. Also, inorder to establish reliable and routine prognostic markers for the canine cutaneous mast cell tumors, other factors of prognostic relevance like staging, recurrence, speed of growth, cytological pleomorphism or differentiation, along with mutations in KIT gene may together be taken into consideration. Grading based on toluidine blue stain score offers promising prognostic inputs in the canine cutaneous mast cell tumors as per our study. Therefore, it can be considered as a point of care test with less turnaround time, cost effective and easier to perform as compared to the immunostaining.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the College of Veterinary Science and Animal Husbandry, DUVASU Mathura, Uttar Pradesh India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our extreme thanks for the indispensable assistance rendered by\u0026nbsp;The Dean (DUVASU), Mathura for official support in providing us with all the necessary facilities during the entire period of this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts of interest with respect to the research, authorship or publication of this research article.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAS- conducted the experimentSNP; planned the design of work and manuscript writingNKG \u0026amp; RS Experimental work planAPS: Manuscript writingSP: surgical resection of tumoursJKC \u0026amp;AK: Statistical analysisKG \u0026amp; YB: Sample collection and experimental workSC \u0026amp; AS: provision of chemicals and manuscript writingAll the authors have equally contributed to this research work\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBae S, Milovancev M, Bartels C, Irvin VL, Tuohy JL, Townsend KL, Leeper H (2020) Histologically low‐grade, yet biologically high‐grade, canine cutaneous mast cell tumors: A systematic review and meta‐analysis of individual participant data. Vet comp oncol 8: 580-589.\u003c/li\u003e\n \u003cli\u003eCamus MS, Priest HL, Koehler JW, Driskell EA, Rakich PM, Ilha MR, Krimer PM (2016). Cytologic criteria for mast cell tumor grading in dogs with evaluation of clinical outcome. Vet pathol 53:1117-1123.\u003c/li\u003e\n \u003cli\u003eCasagrande TAC, Barros LMDO, Fukumasu H, Cogliati B, Chaible LM, Dagli MLZ, Matera JM (2013). The value of molecular expression of KIT and KIT ligand analysed using real-time polymerase chain reaction and immunohistochemistry as a prognostic indicator for canine cutaneous mast cell tumors. Vet Comp Oncol 13: 1\u0026ndash;10.\u003c/li\u003e\n \u003cli\u003eCowell RL, Tyler RD, Meinkoth JH, DeNicola DB. (2007). Diagnostic cytology and hematology of the dog and cat-E-book. Elsevier Health Sciences, Oklahoma.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGiantin M, Vascellari M, Morello EM, et al. (2012). c-KIT messenger RNA and protein expression and mutations in canine cutaneous mast cell tumors: correlations with post-surgical prognosis. J Vet Diagn Invest 24(1):116-126. doi:10.1177/1040638711425945\u003c/li\u003e\n \u003cli\u003eda Costa, R. M. G. (2015). C-kit as a prognostic and therapeutic marker in canine cutaneous mast cell tumours: From laboratory to clinic. The Veterinary Journal, 205(1), 5-10.\u003c/li\u003e\n \u003cli\u003eGrigorev IP, Korzhevskii DE. (2021). Modern Imaging Technologies of Mast Cells for Biology and Medicine (Review). Sovrem Tekhnologii Med.; 13(4):93-107. doi: 10.17691/stm2021.13.4.10. Epub 2021 Aug 28. PMID: 34603768; PMCID: PMC8482833.\u003c/li\u003e\n \u003cli\u003eHahn KA, King GK, Carreras JK. (2004). Efficacy of radiation therapy for incompletely resected grade-III mast cell tumors in dogs: 31 cases (1987\u0026ndash;1998). J. Am. Vet. Med. Assoc. 224: 79\u0026ndash;82.\u003c/li\u003e\n \u003cli\u003eKiupel M, Camus M. (2019). Diagnosis and prognosis of canine cutaneous mast cell tumors. Vet Clinics: Small Anim Pract 49: 819-836.\u003c/li\u003e\n \u003cli\u003eKiupel M, Webster JD, Bailey KL, et al. (2011). Proposal of a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior. Vet Pathol 48: 147-155.\u003c/li\u003e\n \u003cli\u003eMoore AS, Frimberger AE, Taylor D, Sullivan N (2020).\u0026nbsp;Retrospective outcome evaluation for dogs with surgically excised, solitary Kiupel high-grade, cutaneous mast cell tumors. Vet Comp Oncol 18: 402\u0026ndash;408.\u003c/li\u003e\n \u003cli\u003eMullins MN, Dernell WS, Withrow SJ, Ehrhart EJ, Thamm DH, Lana SE (2006). Evaluation of prognostic factors associated with outcome in dogs with multiple cutaneous mast cell tumors treated with surgery with and without adjuvant treatment: 54 cases (1998\u0026ndash;2004). J Am Vet Med Assoc 228: 91-95.\u003c/li\u003e\n \u003cli\u003eMurphy S, Sparkes AH, Brearley MJ, Smith KC, Blunden AS. (2004). Relationships between the histological grade of cutaneous mast cell tumors in dogs, their survival and the efficacy of surgical resection. Vet Rec 154:\u0026nbsp;743-746.\u003c/li\u003e\n \u003cli\u003eNorthrup NC, Howerth EW, Harmon BG et al. (2005). Variation among pathologists in the histologic grading of canine cutaneous mast cell tumors with uniform use of a single grading reference. J of Vet Diagn Invest 17: 561\u0026ndash;\u003c/li\u003e\n \u003cli\u003ePatnaik AK, Ehler WJ, MacEwen EG. (1984). Canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs. Vet Pathol 21(5):469-74. doi: 10.1177/030098588402100503. PMID: 6435301.\u003c/li\u003e\n \u003cli\u003eRibatti D (2018). The staining of mast cells: a historical overview.\u0026nbsp;Int Arch Allergy Appl Immunol 176(1): 55-60.\u003c/li\u003e\n \u003cli\u003eSabattini S \u0026amp; Bettini G (2010). Prognostic value of histologic and immunohistochemical features in feline cutaneous mast cell tumors.\u0026nbsp;Vet Pathol\u0026nbsp;47(4): 643-653.\u003c/li\u003e\n \u003cli\u003eSailasuta A, Ketpun D, Piyaviriyakul P, Theerawatanasirikul S, Theewasutrakul P, Rungsipipat A. (2014). The relevance of cd117-immunocytochemistry staining patterns to mutational exon-11 in c-kit detected by pcr from fine-needle aspirated canine mast cell tumor cells. Vet Med Int 1-8.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eShoop SJ, Marlow S, Church DB, English K, McGreevy PD, Stell AJ, Thomson PC, O\u0026rsquo;Neill DG, Brodbelt DC. (2015). Prevalence and risk factors for mast cell tumors in dogs in England. Canine Genet epidemiol\u0026nbsp;2: 1-10.\u003c/li\u003e\n \u003cli\u003eSridharan G, Shankar AA. Toluidine blue: A review of its chemistry and clinical utility (2012). J Oral Maxillofac Pathol. \u0026nbsp;16(2):251-5. doi: 10.4103/0973-029X.99081. PMID: 22923899; PMCID: PMC3424943.\u003c/li\u003e\n \u003cli\u003eVascellari M, Giantin M, Capello K, Carminato A, Morello EM, Vercelli A, Granato A, Buracco P, Dacasto M, Mutinelli F. (2013). Expression of Ki67, BCL-2, and COX-2 in canine cutaneous mast cell tumors: association with grading and prognosis. Vet pathol 50: 110-121.\u003c/li\u003e\n \u003cli\u003eVillamil JA, Henry CJ, Bryan JN, Ellersieck M, Schultz L, Tyler JW, Hahn AW. (2011). Identification of the most common cutaneous neoplasms in dogs and evaluation of breed and age distributions for selected neoplasms. J Am Vet Med Assoc 239: 960-965.\u003c/li\u003e\n \u003cli\u003eWebster JD, Yuzbasiyan-Gurkan V, Kaneene JB, Miller R, Resau JH, Kiupel M. (2006). The role of c-KIT in tumorigenesis: evaluation in canine cutaneous mast cell tumors. Neoplasia 8(2):104-11. doi: 10.1593/neo.05622. PMID: 16611403; PMCID: PMC1578516.\u003c/li\u003e\n \u003cli\u003eWeisse C, Shofer FS, Sorenmo K. (2002). Recurrence rates and sites for grade II canine cutaneous mast cell tumors following complete surgical excision. J Am Anim Hosp Assoc 38: 71\u0026ndash;3.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cutaneous mast cell tumors, canine, immunocytochemistry, grading","lastPublishedDoi":"10.21203/rs.3.rs-3881523/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3881523/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of the study was to characterize the canine cutaneous mast cell tumor based on cytomorphology and to assess the significance of cytology, histology and immunohistochemistry in its diagnosis and prognosis. The tumors were cytologically and/or histologically graded as high or low. The tumors were also graded based on the cytoplasmic granularity by toluidine blue staining method and immunostaining pattern shown by cKIT. A comparison of various parameters like breed, age, sex, tumor location size, granulation, cytological/histological grade, cKIT staining pattern and cKIT IS between the dead and live animals was made. Based on histology/cytology, 12 tumors were of high grade and four were of low grade. Median survival time for the high grade tumor was found to be 5 months. Immunocytochemistry revealed intense cytoplasmic diffuse pattern followed by stippled and membraneous patterns. No significant difference was noted for tumor location, size, sex, histological grade, cKIT Immunostaining pattern and IS between the two prognostic groups dead and live. The KIT staining pattern was not much informative for the prognosis of canine cutaneous mast cell tumors in this study. But a significant difference between the two groups was noted for the cytoplasmic granularity demonstrated by toluidine blue stain. The toluidine blue staining score served as better prognostic indicator with poor granulation favouring a worse prognosis. Therefore, toluidine blue staining may be considered a cost effective, easier and rapid method to perform as compared to immunostaining in the prognosis of canine cutaneous mast cell tumour.\u003c/p\u003e","manuscriptTitle":"High Prognostic relevance of toluidine blue stain score in canine cutaneous mast cell tumor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-24 09:35:49","doi":"10.21203/rs.3.rs-3881523/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a994c850-9a5f-46a4-b7bb-b25f3e1bca1c","owner":[],"postedDate":"January 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-23T00:29:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-24 09:35:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3881523","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3881523","identity":"rs-3881523","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}