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Vet Clin Pathol. 2022;00:1–10. | 1wileyonlinelibrary.com/journal/vcp Received: 13 July 2021 | Revised: 3 November 2021 | Accepted: 11 November 2021 DOI: 10.1111/vcp.13098 O R I G I N A L A R T I C L E Inclusion of fibroblasts and collagen fibrils in the cytologic grading of canine cutaneous mast cell tumors Paulo R. O. Paes | Rodrigo S. Horta | Ludimila C. Luza | Felipe Pierezan | Mariana P. Costa | Gleidice E. Lavalle © 2022 American Society for Veterinary Clinical Pathology Department of Veterinary Medicine and Surgery, Veterinary School, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil Correspondence Paulo R. O. Paes, Universidade Federal de Minas Gerais (UFMG), 6627 Antônio Carlos Avenue, Belo Horizonte, MG 31270- 901, Brazil. Email: paulopaes@vet.ufmg.br Funding information National Council for Scientific and Technological Development Abstract Background: Fibroblasts and/or collagen fibrils have not been included in previous cytologic grading schemes of canine mast cell tumors (MCTs), and their association with biological behavior is broadly debated. Objectives: This study aimed to evaluate the cytologic findings of canine MCT, with emphasis on the microenvironment, and propose a novel cytologic grading system correlated with mortality and histologic grade. Material and Methods: Cytology smears of canine cutaneous MCTs were retrospec- tively reviewed and compared with their histopathologic counterparts using Cohen´s Kappa test. One- year survival rates were also compared with the cytologic and histo- pathologic variables using Pearson´s correlation test. Results: From 92 first- occurrence canine cutaneous MCTs, the five features most as- sociated with mortality were selected for a new grading system. The five features were cytoplasmic granulation, fibroblast and/or collagen fibril concentrations, and the presence of mitotic figures, multinucleation, and karyomegaly. Among concord- ant histopathologic and cytologic cases (ie, the same grades using both systems), mor- tality rates were 2.6% (1/38) for low- grade and 71.4% (10/14) for high- grade cases (P < 0.001, chi- square). For false- negative and false- positive results, mortality rates were 33% (1/3) and 45% (5/11), respectively (P = 0.707). Conclusions: Unlike the Camus cytologic grading system, the present amendment ex- cluded binucleation and included fibroblasts and/ or collagen fibrils, which in higher concentrations were associated with increased survival and a low histopathologic grade. Cytologic grading with the inclusion of fibroblast and collagen fibril concentra- tions correlated with survival, as did the Camus cytologic and Kiupel histopathologic grades; however, further studies are needed to confirm the prognostic value of this novel cytologic grading scheme. K E Y W O R D S cancer, dog, granulation, microscopy, mitotic figures, oncology www.wileyonlinelibrary.com/journal/vcp mailto: https://orcid.org/0000-0002-1366-7352 https://orcid.org/0000-0002-9885-4498 https://orcid.org/0000-0002-3319-8784 https://orcid.org/0000-0002-2701-9954 mailto:paulopaes@vet.ufmg.br 2 | PAES Et Al. Mast cell tumor (MCT) is a common malignancy in dogs, occurring mainly as solitary skin nodules, although the cutaneous presentation must be differentiated from the subcutaneous one since the latter is reported to have less aggressive behavior.1 As it presents a wide variety of mac- roscopic presentations, canine MCT can mimic other skin tumors and even nonneoplastic lesions.2,3However, as most neoplastic mast cells resemble their nonneoplastic counterparts, canine MCTs are usually easily identified by means of the cytologic examination of fine- needle aspirates (FNAs),4 with a sensitivity that might be as high as 96%.5 Although it rarely represents a diagnostic challenge, the bio- logical behavior of canine cutaneous MCT is highly variable, and veterinary pathologists, clinicians, and surgeons are interested in identifying the most aggressive cases to guide therapy.5Currently, in an attempt to predict its behavior, it is common to grade canine cutaneous MCT in two histopathologic systems.2,6,7In the Patnaik three- tier system,8 dogs with grade I and III MCTs present the high- est and lowest life expectancy, respectively. In a more objective two- tier system, proposed by Kiupel et al, high- grade MCTs have a worse prognosis than low- grade MCTs9. Despite the high diagnostic sensitivity of cytologic examinations, the use of such a technique for canine MCT grading is questionable, which results in difficulties in establishing the appropriate therapeu- tic approach without surgical biopsy.4,5,10– 13 When the Kiupel grading system9 was adapted for cytologic grading, Hergt et al12 and Scarpa et al13 noted “false- negative” results in 2/38 (5%) and 5/105 (4.8%) of cytologically low- grade MCTs, respectively. False negatives are con- sidered cytologically low- grade cases that are histologically high grade, while false positives are cytologically high grade but histologically low- grade cases. False negatives can be more harmful to clinical- surgical conduct than false positives since the latter tends to lead to more cau- tious planning.11However, when using poor cytoplasmic granulation or the presence of at least two other specific cytologic features for cyto- logic classification as high grade, Camus et al11 reported 15/22 (32%) false positives and only 2/130 (1%) false negatives. Fibroblasts and/or collagen fibrils have not been included in the cytologic grading of canine MCT.11– 13Nevertheless, those features are commonly observed in MCTs, especially well- differentiated tumors, but their association with biological behavior is debated and remains unknown.14 The aim of this study was to evaluate the cytologic find- ings of first- occurrence canine cutaneous MCT, including those al- ready established in the literature, but with additional consideration of the microenvironment (specifically, fibroblasts and collagen fibrils), to propose a novel cytologic grading system, and evaluate its correlation with mortality and the Kiupel histopathologic grade. 1 | MATERIAL AND METHODS 1.1 | Study design A retrospective study of first- occurrence canine cutaneous MCTs was performed in the Veterinary School Hospital of Universidade Federal de Minas Gerais (HV- UFMG), Belo Horizonte, Brazil, between July 2014 and January 2020. Inclusion criteria were dogs with a histopathologic diagnosis of cutaneous MCT and no history of previous MCT, availability of cytologic smears and histologic sec- tions, and curative- intent surgical excision performed at the insti- tution. Histopathologic examinations performed more than 90 days after the cytologic examination were excluded, as were cases with cytologic samples showing less than 100 intact mast cells. The in- cluded dogs were divided according to the number of MCTs (one, two, or more than two) and according to the largest tumor size (smaller or larger than 4 cm) as performed in other studies.7Clinical staging was performed according to the World Health Organization (WHO), through clinical examination, abdominal ultrasound, and when enlarged, lymph node (LN) assessment through cytology and/ or histopathology examination. 1.2 | Cytologic and histopathologic analysis Cytologic samples were obtained by well- trained clinicians using a 25×7 needle, with three to five slides; the first samples were ob- tained without aspiration to reduce blood contamination, then, they were obtained with aspiration. Cytologic examinations were per- formed on samples collected in the hospital by FNA and stained with Diff- Quik (Romanowsky) stain. The Camus grading system3 was applied to all cytologic samples. MCTs were considered high grade if there was poor granulation or at least two of the following features: binucleated or multinucleated cells, mitotic figures, and karyomegaly. Those findings are wellillus- trated in the paper of Camus et al.11 The cytologic variables evaluated and their definitions are sum- marized in Table 1. Using 40× and 100× magnification (Field num- ber [FN] 22 eyepiece, 430 μm) microscopy, the cellularity and mast cell disposition were analyzed. For cellularity evaluations, samples with several fields filled with mast cells were classified as high cel- lularity (Figures 1– 3), samples with several healthy mast cells close to the minimum required (100– 200 cells) were considered as low cellularity, and those in- between were considered as intermediate cellularity. The samples were classified according to the proportion of clustered cells in relation to free cells, with the results presented as the percentage of clustered cells (Figures 1- 3). For this, in the presence of large or medium mast cell clusters, the mast cells were considered as “grouped.” At 100× and 400× magnification, cyto- plasmic granulation was also evaluated (Figure 4, Figures S1- S8). Multinucleation and mitotic figures were also present, as seen in Figure 4. Fibroblast, collagen fibril, eosinophil, and neutrophil numbers were evaluated at 1000× and 400× magnification and expressed as the number per field. Fibroblasts and collagen fibrils were counted together. All spindle- shaped cells were considered fibroblasts. The presence of several fibroblasts and/or collagen fibrils in some or several fields (1000×) was considered a high concentration (Figures 5- 6), while the presence of a few fibroblasts and/or collagen fi- brils in some or several fields (1000×) was considered a moderate | 3PAES Et Al. concentration (Figures 7- 8). The presence of rare (1 or 2) fibroblasts and/or collagen fibrils was considered low concentration (1000×) (Figures 9- 10). In samples with low cellularity, the lower proportion of mast cells associated with the presence of rare fibroblasts and/or collagen fibrils resulted in the classification of a moderate concen- tration of fibroblasts and/or collagen fibrils. F I G U R E S 1- 4 Mast cell tumor, dog, skin. Diff- Quik. Figures 1- 2. Highly clustered mast cells at 40× magnification. Clustered mast cells represent 90% (Figure 1) and 60% (Figure 2) of the neoplastic cell population. In Figure 2, only the cells on the right were considered clustered (arrow). Figure 3. Poorly clustered mast cells (10% clustered cells), 40×. Figure 4. Poorly granulated mast cells, multinucleation (arrowhead) and mitotic figure (arrow), 400× TA B L E 1 Criteria applied for cytologic grading canine mast cell tumors Variable (magnification) Criterion Cellularity (40 and/or 100×) High: cells cover 1/3 of the slide Intermediate Low (approximately 100– 200 cells per slide) Proportion of clustered cells (40 and/or 100×) ≥40% ≤20% Concentration of fibroblasts and/or collagen fibrils (100× and 400×) Intermediate to high (moderate to intense concentration in at least some areas of the slide) Low/absent Concentration of eosinophils and/or neutrophils (100× and 400×) Intermediate to high (moderate to intense concentration in at least some areas of the slide) Poor (scarce to absent) Cytoplasmic granulation of mast cells (100× and 400×) High Poor (moderate to absent and variable) Binucleated and multinucleated mast cells, anisocytosis, mitotic figures, karyomegaly (100× and 400×) Present Absent Note: Cytologic high- grade mast cell tumors presented poor granulation or, in the presence of high cytoplasmic granulation, at least two of the following four criteria: presence of multinucleated cells, mitotic figures, karyomegaly, and low/absent concentration of fibroblasts and/or collagen fibrils. 4 | PAES Et Al. Histopathologic examination was performed at three labora- tories, including the Laboratory of Veterinary Anatomic Pathology from UFMG, Belo Horizonte- MG, Laboratory of Comparative Pathology from UFMG, Belo Horizonte- MG; and VETPAT Laboratory, Campinas- SP. The Kiupel grading system9 was applied to all histopathologic analyses. It was not an objective of this study to evaluate the surgical margins, and trimming was not standardized. 1.3 | One- year survival rate The 1- year survival rate was assessed through clinical follow- up, as recorded in the data system of the Veterinary School Hospital of UFMG, and, when necessary, via telephone contact with the own- ers of the animals involved in the study. The course of the disease was investigated between the cytologic diagnosis and the following F I G U R E S 5 - 1 0 Mast cell tumor, dog, skin. Diff- Quik. Figures 5- 6. High fibroblast and collagen fibril concentrations. At ×10 objective, the field shows several mast cells, and it is filled with collagen fibrils (pink- staining material) and fibroblasts (fusiform cells with basophilic cytoplasm) (Figure 5). The same slide at ×40 objective and more details of mast cells, collagen fibrils (arrow), and fibroblasts (arrowhead) (Figure 6). Figures 7– 8. Intermediate fibroblast and collagen fibril concentrations. At ×10 objective, note rare collagen fibrils (arrow) and fibroblasts (arrowhead) among high concentrations of mast cells (Figure 7). The same slide at ×40 objective showing more detail of the mast cells, collagen fibrils (arrow), and fibroblasts (arrowhead) (Figure 8). This feature is repeated in several fields of this same slide resulting in classification as intermediate concentration. Figures 9– 10. Low fibroblast and collagen fibril concentrations. At ×10 objective, note rare collagen fibrils (arrow) and fibroblasts (arrowhead) among a high concentration of mast cells (Figure 9). The same slide at 400× and more details of mast cells, collagen fibrils (arrow), and fibroblasts (arrowhead) (Figure 10). Such findings were presented in only one more field of this same slide, which resulted in the classification of low concentration | 5PAES Et Al. 365 days, evaluating the outcome and cause of death. For dogs with more than one nodule classified in different histopathologic grades, those with higher grades were considered. Animals that died in less than 365 days due to an unknown cause or a reason different from the neoplasm were excluded from the study on life span. Mortality was determined by the percentage of deaths within 1 year. 1.4 | Statistics Inferential statistics were performed using GraphPadPrism v. 6.02 software with a significant index of 5%. Pearson’s test was used to estimate the correlation of the cytologic variables with the 1- year survival rate and Kiupel histopathologic grade. The significant cor- relations were considered weak (r < 0.30), moderate (0.30 ≤ r < 0.7), or strong (r ≥ 0.70), and the five cytologic features most correlated with the 1- year survival rate were selected for a new cytologic grad- ing scheme. The chi- square test was used to assess the frequency dispersion of each variable in relation to mortality. Cohen´s kappa test was used to evaluate the statistical relationship between both cytologic grading schemes and those with the Kiupel grading sys- tem. The kappa value was indicative of no agreement (κ < 0), slight (0.01 < κ < 0.20), fair (0.21 < κ < 0.40), moderate (0.41 < κ < 0.60), substantial (0.61 < κ < 0.80), or almost perfect (0.81 < κ < 1.00) agreement. 2 | RESULTS A total of 121 cases of first- occurrence canine MCT met the initial search criteria. Of these, 23 were excluded because a histopatho- logic section was not available and six because the diagnoses were classified as subcutaneous MCT. Thus, 92 cases were included in the study. Regional LNs were assessed, when enlarged at palpation, in 29 dogs by histology (n = 25; of which 13 had metastasis), cytology (n = 2, with possible metastasis, but LN was not removed), or both (n = 2; of which one had metastasis). Two dogs had more than three nodules, and the LNs were not evaluated in these cases. According to the WHO, there was no stage IV disease, two- stage III, and the remainingwere stage I or II, depending on the LN status (overt me- tastasis in 14 cases and no metastatic in 13 cases). Of the 92 cases, 66 had information about the 1- year survival (six were obtained through telephone contact and the remaining through clinical evaluation). Additional tests were performed according to the clinician ś discretion. Seventeen of the 66 (26%) dogs died from MCT- related causes, of which eight were euthanized, and nine died of nat- ural causes. Euthanasia and death were both related to progressive disease (ie, local tumor recurrence, or LN or distant metastasis). The included dogs were 9.6 ± 3.5 years old (mean ± SD) at diag- nosis. Of the 92 cases, there were 32 mixed breed dogs, 13 Labrador retrievers, 10 Boxers, six American Pit- Bull terriers, six Golden re- trievers, three Pinschers, two Basset hounds, two Lhasa Apsos, two Malteses, two Poodles, two Pugs, and 12 of different breeds. Of the 92 cases, 78 had solitary lesions, 10 had two nodules, and four had more than two nodules. The nodules were in the trunk (n = 41), limbs (28), head (5), scrotum (4), prepuce (4), vulva (2), or more than one of these regions (8). MCT- related mortality was not statistically differ- ent (P = 0.507) for cases with a single lesion (14/57), two nodules (1/5), or more than two lesions (2/4). Tumor size was not available for 13 cases. Nodules of up to 4 cm were identified in 60/79 cases, and 19/79 had lesions larger than 4 cm. The MCT- specific mortality of 9/12 (75%) was significantly higher in cases with lesions larger than 4 cm, compared with 6/40 (15%) mortality in cases with lesions smaller than 4 cm (P < 0.001, chi- square). TA B L E 2 Pearson´s correlations between the cytologic features and 1- year survival rate in 92 first- occurrence canine cutaneous mast cell tumors Variable P- value rP value Cellularity P = 0.519 rP = 0.081 Proportion of clustered cells P = 0.002 rP = 0.377 Concentration of fibroblasts P < 0.001 rP = 0.508 Concentration of collagen fibrils P = 0.020 rP = 0.286 Concentration of fibroblasts/collagen fibrils P < 0.001 rP = 0.561 Concentration of eosinophils P = 0.331 rP = −0.122 Concentration of neutrophils P = 0.379 rP = 0.110 Cytoplasmic granulation P < 0.001 rP = 0.590 Anisocytosis P = 0.082 rP = 0.215 Binucleation P = 0.017 rP = 0.293 Multinucleation P < 0.001 rP = 0.494 Karyomegaly P < 0.001 rP = 0.449 Mitotic figures P < 0.001 rP = 0.522 rP, Pearson correlation coefficient. For significant P- value (P < 0.05), correlations are considered weak (rP <0.3), moderate (0.3 < rP <0.7) or strong (rP >0.7). 6 | PAES Et Al. All 92 dogs had their MCT surgically removed. Additionally, che- motherapeutic drugs were used in 32/92 cases, including vinblastine in 12, lomustine in 11, chlorambucil in two, or a combination of these in seven cases. Table 2 shows the statistical relationship (Pearson ś test) between the cytologic variables and 1- year survival rates. In decreasing order, the 1- year survival rate was moderately correlated with cytoplasmic granulation, fibroblast and/or collagen fibril concentrations, mitotic figures, multinucleation, karyomegaly, and the proportion of clustered cells and was weakly correlated with binucleation. Mortality was de- termined for each cytologic criterion for the study of frequency dis- persion (chi- square) (Table 3). The five cytologic features with the highest Pearson correlation values (1- year survival rate) were selected for a new cytologic grading scheme. As proposed by Camus,3 the presence of predominantly poor granulation was used as a criterion for high- grade. In the absence of poor granulation, at least two of the following criteria were needed to determine high- grade: low/absent fibroblast or collagen fibril concentrations, and/or the presence of mi- totic figures, multinucleated cells, and karyomegaly. The 1- year survival rate was moderately correlated with cyto- logic grading by the Camus scheme (P < 0,001; rP = 0.504), and by that of the present study (P < 0.001; rP = 0.695), as well as with the Kiupel grading system (P < 0.001; rP = 0.507). The statistical relationship of the cytologic variables and the Kiupel grade was assessed (Table 4). In decreasing order, significant correlations were observed for karyomegaly, cytoplasmic granula- tion, multinucleation, mitotic figures, the proportion of clustered cells, concentration of fibroblasts and/or collagen fibrils, and cel- lularity. There was a moderate correlation between fibroblasts and collagen fibrils (P < 0.001, rP = 0.629). Despite not having a correla- tion with the Kiupel grade, binucleation was moderately correlated with karyomegaly (P < 0.001, rP = 0.649) and multinucleation (P < 0.001, rP = 0.443), and weakly correlated with fibroblast and/ or collagen fibril concentrations (P = 0.008,rP = 0.277), cytoplasmic granulation (P = 0.008, rP = 0.277), and the presence of mitotic fig- ures (P = 0.042, rP = 0.212). There was substantial agreement between the cytologic grad- ing systems of Camus and the present study (κ = 0.773, SE: 0.067, Cohen´s Kappa test). Kiupel grading had fair agreement with the cy- tologic grading of Camus (κ = 0.336; SE: 0.096) and a higher mod- erate agreement with the cytologic grading of the present study (κ = 0.472; SE: 0.096). TA B L E 3 Cytologic features of 92 first- occurrence canine cutaneous mast cell tumors Variable Concentration Cases Mortality Significance (χ2) Cellularity High 54/92 (59%) 12/42 (29%) P = 0.437 Intermediate 18/92 (19%) 33/9 (33%) Low 20/92 (22%) 2/15 (13%) Clustered cells ≥40% 58/92 (63%) 5/40 (15%) P = 0.002 ≤20% 34/92 (37%) 12/26 (46%) Collagen fibrils High 80/92 (59%) 4/35 (11%) P = 0.005 Low/absent 38/92 (41%) 13/31 (42%) Fibroblasts High 67/92 (73%) 6/46 (13%) P < 0.001 Low/absent 25/92 (27%) 11/20 (55%) Fibroblasts/Collagen Fibrils High 69/92 (75%) 6/48 (13%) P < 0.001 Low/absent 23/92 (25%) 11/18 (61%) Neutrophils High 18/92 (20%) 1/9 (11%) P = 0.280 Low/absent 74/92 (80%) 16/57 (28%) Eosinophils High 27/92 (29%) 3/20 (15%) P = 0.188 Low/absent 65/92 (71%) 14/46 (30%) Cytoplasmic granulation High 69/92 (75%) 6/49 (12%) P < 0.001 Poor 23/92 (25%) 11/17 (65%) Binucleation Absent 50/92 (54%) 5/31 (16%) P = 0.021 Present 42/92 (46%) 12/35 (34%) Multinucleation Absent 79/92 (86%) 9/54 (17%) P = 0.003 Present 13/92 (14%) 8/12 (67%) Karyomegaly Absent 54/92 (59%) 3/36 (8%) P < 0.001 Present 38/92 (41%) 14/30 (47%) Mitotic figures Absent 83/92 (90%) 10/57 (18%) P < 0.001 Present 9/92 (10%) 7/9 (78%) | 7PAES Et Al. Of the 92 cases, the number determined to be of low grade was 53 (57%) using the system of Camus, 59 (64%) using the system of the present study, and 68 (74%) using the Kiupel histologic grad- ing system (Table 5). When the results of the Kiupel grading system and the present cytologic grading scheme were paired, 6/59 cases (10%) were cytologically low grade and histologically high grade (false negatives), while 15/33 (46%) were cytologically high grade and histologically low grade (false positives). Among concordant histopathologic and cytologic cases (ie, the same grade using both systems), mortality rates were 1/38 (2.6%) for low- grade and 10/14 (71.4%) for high- grade cases (P < 0.001, chi- square). For the cytol- ogies with false negatives and false positives, mortality rates were 1/3 (33%) and 5/11 (45%), respectively (P = 0.707). 3 | DISCUSSION The cytologic grading scheme for canine MCT proposed by Camus et al11 has gained popularity since it was published and is currently used by many pathologists. In that study, the cytologic findings showed that the highest correlation with mortality was poor cyto- plasmic granulation, the presence of karyomegaly, multinucleation, binucleation, and mitotic figures, while nuclear pleomorphism and the concentration of collagen fibrils showed a weak correlation with mortality. Hergt12 and Scarpa13 also observed a weak correlationbetween nuclear pleomorphism (bizarre nucleus) and tumor aggres- siveness. According to Camus et al, the flattening of the nucleus during the making of the cytologic smear may mischaracterize the indentations, and lobulations observed histologically, which might prevent the observation of nuclear pleomorphism.11 The present study partially supports those findings since the features most as- sociated with mortality were, in decreasing order, poor cytoplasmic granulation, a low concentration of fibroblasts and/or collagen fibrils, the presence of mitotic figures, multinucleation, and karyomegaly. Poor cytoplasmic granulation was considered a major criterion for high- grade cases, and in the absence of this, the presence of at least two of the other four cytologic criteria was required to consider a tumor high- grade cytologically. The proportion of clustered cells and binucleation were also correlated with the 1- year survival rate, but since these correlations were weaker, they were not included as cri- teria for the novel cytologic grading system. Thus, the main changes related to the system proposed by Camus et al were (a) including a low concentration of fibroblasts and/or collagen fibrils as a criterion for high grade, and (b) excluding binucleation as a grading criterion. However, although binucleation was not used as a criterion for the proposed grading system, its observation is an important alert for TA B L E 4 Pearson´s correlations between cytologic features and Kiupel grade in 92 first- occurrence canine cutaneous mast cell tumors Variable P- value rP value Cellularity P = 0.039 rP = 0.216 Proportion of clustered cells P = 0.002 rP = 0.314 Concentration of fibroblasts P = 0.065 rP = 0.194 Concentration of collagen fibrils P = 0.320 rP = 0.105 Concentration of fibroblasts/collagen fibrils P = 0.028 rP = 0.229 Concentration of eosinophils P = 0.313 rP = 0.106 Concentration of neutrophils P = 0.857 rP = −0.019 Cytoplasmic granulation P < 0.001 rP = 0.400 Anisocytosis P = 0.431 rP = 0.083 Binucleation P = 0.150 rP = 0.151 Multinucleation P < 0.001 rP = 0.399 Karyomegaly P < 0.001 rP = 0.407 Mitotic figures P < 0.001 rP = 0.388 TA B L E 5 Mortality of first- occurrence canine cutaneous mast cell tumors, according to the cytologic grading system of the present study and of Camus, and histologic grading system according to Kiupel System Grade Cases Mortality Significance (χ2) Present study Low grade 59/92 (64%) 2/41 (5%) P < 0.001 High grade 33/92 (36%) 15/25 (60%) Camus et al (2016) Low grade 53/92 (58%) 3/35 (9%) P < 0.001 High grade 39/92 (42%) 14/31 (45%) Kiupel et al (2011) Low grade 68/92 (74%) 6/49 (12%) P < 0.001 High grade 24/92 (26%) 11/17 (65%) 8 | PAES Et Al. the presence of other cytologic features in canine MCT samples since this finding was moderately correlated with karyomegaly and multinucleation. Similarly, the cellularity and percentage of clus- tered cells were not used as criteria for cytologic grading, but they did correlate with the Kiupel histopathologic grade. Poorly cellular cytologic samples tended to correlate with low- grade tumors. This result indicates that the number of cells collected in the FNA is not only associated with factors such as the location of the tumor and the capability of the veterinarian who collected the sample. Also, most MCT samples with at least 40% clustered cells had lower mor- tality compared with those that had a higher concentration of free (dissociated) cells. The increased number of clustered cells was as- sociated with histologically low- grade tumors. Mesenchymal cells have no intercellular adhesions, and their neoplasms usually show free (dissociated) cells on cytology, especially for round cell tumors. However, with the effect of the extracellular matrix, these cells may be loosely grouped.15 The moderate correlation between the low concentration of col- lagen fibrils and mortality observed in the present study differs from the weak correlation between these variables reported by Camus et al11 possibly due to the methodologies applied. Camus et al con- sidered collagen fibrils only as present or absent.11 In the present study, most samples showed significant amounts of collagen, char- acterized by the presence of a moderate concentration of these fibrils in some areas of the slide, and these cases had significantly lower mortality rates than those with absent or low collagen fibril concentrations. Although the concentration of fibroblasts was not investigated in cytologic samples of canine MCT in other studies,11– 13 these cells are frequently seen in canine MCT, especially in the well- differentiated ones.14Based on results obtained in the present study, the concen- tration of fibroblasts might be relevant for the prognosis dogs with cutaneous MCTs, since samples that had areas with a moderate con- centration of fibroblasts were associated with lower mortality than those with absent or low concentrations of fibroblasts. Although fibroblasts could not be differentiated from other spindle- shaped mesenchymal cells without the support of special stains or immunohistochemistry, they represent the main dermal spindle cell type.16When activated, these cells can have features suggesting atypia, such as anisokaryosis, binucleation, and promi- nent nucleoli.14 Almost all the fibroblasts observed in the present study were oval- to- spindle shaped, often with prominent nucleoli and occasionally with binucleation. These characteristics are typi- cal of activated fibroblasts.14 Since a moderate correlation was ob- served between fibroblast and collagen fibril concentrations; they were used together in the grading system of the present study. However, it is not possible to establish the origin of fibroblasts in cy- tologic smears, which might be relevant to understanding their role in cancer progression. The differentiation between normally activated fibroblasts and cancer- associated fibroblasts can only be performed through immunohistochemistry.17Cancer- associated fibroblasts partic- ipate in all phases of tumor progression, including metastasis through the production of potent growth factors.17,18 In contrast, normal, activated fibroblasts might be important in inhibiting tumor growth by reversing some of the substances produced by cancer- associated fibroblasts, such as tumor growth factor- beta (TGF- beta).17Cancer- associated fibroblasts express the fibroblast- activating protein, absent in resident fibroblasts, except those involved in the formation of granulation tissue and chronic in- flammatory processes, such as liver cirrhosis.17,18 The expression of fibroblast- activating protein in canine MCTs has been shown to have a positive correlation with the Patnaik and Kiupel grading systems, mitotic index, and Ki- 67 immunolabeling, suggesting a negative impact of cancer- associated fibroblasts on prognosis.18 In feline squamous cell carcinoma, a positive correlation between the number of cancer- associated fibroblasts and shorter survival times has been reported.19 However, according to Kalluri17, even though cancer- associated fibroblasts have been considered auxil- iary for tumor expansion for decades, it is currently believed that these cells may also act as negative regulators of cancer growth. One study correlated the infiltration of mast cells in the liver and spleen with shortened survival times. Nevertheless, the cy- tology was considered negative for neoplastic infiltration if the sample did not contain any mast cells, if it contained rare, scat- tered, individualized, well- granulated mast cells. or if it contained well- differentiated mast cells associated with connective tissue elements, which might indicate fibrosis and a potential role of mast cells in such events.20 There was high agreement between the three grading schemes applied to canine cutaneous MCTs. Both cytologic grading systems and the Kiupelhistopathologic system could predict 1- year survival rates using univariate analysis. There was an agreement of 71/92 (77%) between the cytologic grading scheme of the present study and the Kiupel histopathologic grade. Specifically,15/33 cases (45%) were cytologically high grade but histologically low grade, while 6/59 cases (10%) were cytologically low- grade but histologically high grade. These values were higher than the 31.8% false- positive cases and 1.5% false- negative cases reported by Camus et al.11In spite of that, the newly proposed cytologic scheme was better correlated with mortality (P < 0.001; rP = 0.504 for Camus and rP = 0.695 for the newly proposed scheme). Among concordant histopathologic and cytologic cases (ie, the same grade using both systems), there was one death among 38 low- grade cases (2.6%) and 10 among 14, for high grade (71.4%) (P < 0.001, chi- square). In 11 false- positive cytology cases, there were five MCT- related deaths (45%) and, despite the small number of samples, it is possible to suggest that cytologic grade could pres- ent a prognostic value even after knowledge of the histopathologic grade. However, the mortality rate of 1/3 (33%) on false- negative cytology cases was deemed too small, and new studies must be per- formed on this subject. When comparing cytologic samples stained with two differ- ent dyes, Sabattini et al observed that although the use of Diff- Quik resulted in less granulation in 18% of samples also stained with May- Grünwald- Giemsa, the grades obtained were similarly | 9PAES Et Al. correlated with the histopathologic grade.6 One study recom- mended the fixation of samples with methanol at least 5 min- utes before using dyes to avoid the poor staining of granules.4 Pathologists must be aware that poor staining should not be mis- interpreted as poor cytoplasmic granulation. In the present study, as the slides were already stained, it was not possible to compare the effects of fixation or compare Diff- Quik with May- Grünwald- Giemsa staining. Poor cell granulation is a major criterion for cytologic grading, which is difficult to standardize; this subject is illustrated in Figures S1- S8. Diff- Quick failure was frequently observed among the cytologic MCT slides used in this and other studies. These preparations were not included in this study be- cause they did not pass the inclusion criteria. In these samples, a reduced dye impregnation was noted in loose cells more than clustered cells. Reduced impregnation in larger areas occurred only occasionally (Figures S5 and S6). Among samples without any stain, all had evident morphologic abnormalities, such as multinu- cleation, karyomegaly, and mitotic figures, which made us believe that this was not a failure of the dye, but rather true poor granula- tion (Figures 4 and S8). In two cases, less than 2% of the samples evaluated, there was a doubt whether there was poor granula- tion or dye failure. Those samples were excluded for not having a histopathologic counterpart, but they were not completely free of stained granules, as seen in Figures 4 and S8. Diff- Quick is routinely used in the laboratory and teaching veterinary hospi- tal of our university, which is also useful for enabling students to perform the cytologic exam in veterinary clinics. However, the results found here may differ in some points made with other Romanowsky dyes, which is, therefore, a limitation of this study. As for any cytologic grading system, it must be considered the possible impact of subjective evaluation for some criteria, as cyto- plasmic grading, as well as different techniques used during sam- ple collection (ie, with or without aspiration) and quality of the smears made by clinicians with different expertise. This was a retrospective study. Despite current LN assessment recommendations for cytology and histology in cases of MCT, as recommended for liver and splenic aspirates, LN assessments were not routinely performed, resulting in incomplete staging. These were recognized as limitations of the study along with the use of different laboratories and pathologists for the histopathologic analyses and assessments using the Kiupel grade. As for clinical and retrospective research, there are several other variables, such as completeness of excision and neoadjuvant and adjuvant therapies, which made anal- ysis by means of multivariate statistics impossible. The authors also recognize that a clinical follow- up of 1 year might be insufficient, as some MCT cases may take longer to progress and relapse. In a subset of cases, the cause of death could not be confirmed as some dogs were not clinically monitored on a regular basis and were not subjected to postmortem examination. However, this study supports the results obtained by Camus et al11 while also demonstrating the importance of the microenvi- ronment and the association of fibroblasts and/or collagen fibrils with lower grades of malignancy. Survival was equally correlated with the Kiupel histopathologic grade, Camus cytologic scheme, and the present amendment with the inclusion of the microenvironment. However, further studies with completely staged dogs, longer fol- low- up times, and differently trained clinicians and pathologists are needed to evaluate agreement in the application of this grading sys- tem, as well as to confirm its prognostic value. ACKNOWLEDG MENTS National Council for Scientific and Technological Development (CNPq), Minas Gerais State Research Support Foundation (FAPEMIG). DISCLOSURE The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article. ORCID Paulo R. O. Paes https://orcid.org/0000-0002-1366-7352 Rodrigo S. Horta https://orcid.org/0000-0002-9885-4498 Felipe Pierezan https://orcid.org/0000-0002-3319-8784 Gleidice E. Lavalle https://orcid.org/0000-0002-2701-9954 R E FE R E N C E S 1. Kiupel M. Mast cell tumor. In: Meuten DJ, ed. Tumors in Domestic Animals. 5th ed. John Wiley and Sons; 2017:176- 202. 2. Strefezzi RF, Kleeb SR, Xavier JG, Catão- Dias JL. Prognostic indica- tors for mast cell tumors. Brazil J Vet Pathol. 2009;2:110- 121. 3. Welle MM, Bley CR, Horward J, Rüfenacht S. Canine mast cell tu- mours: a review of the pathogenesis, clinical features, pathology and treatment. Vet Dermatol. 2008;19(6):321- 339. 4. Kiupel M, Camus M. 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