Gene Expression and Immunohistochemical Analyses of c-Myc in Canine and Feline Soft Tissue Fibrosarcomas

TL;DR

This study examines c-Myc gene and protein expression in dog and cat fibrosarcomas, finding higher levels in more aggressive tumors, suggesting a potential role in prognosis.

Contribution

The study is the first to correlate c-Myc expression with histological grading in canine and feline fibrosarcomas, extending human cancer research to veterinary oncology.

Findings

c-Myc protein was positive in 75% of grade II and all grade III fibrosarcomas in dogs and cats.

c-Myc gene expression was up-regulated in grade III compared to lower-grade fibrosarcomas.

Higher c-Myc levels correlated with increased tumor aggressiveness as per histological grading.

Abstract

Cellular myelocytomatosis (c-Myc) has been recommended as a prognostic marker in soft tissue fibrosarcoma in humans. The aim of our study was to assess the mRNA and protein expression of c-Myc in canine and feline fibrosarcoma samples and correlate their expression to histological grading degrees. The expression of the c-Myc marker was significantly correlated to an increased abnormality of cell appearance in canine and feline fibrosarcoma samples. Additional studies are essential to examine the therapeutic and prognostic role of c-Myc in canine and feline fibrosarcoma.

Canine and feline fibrosarcomas are malignant tumors of mesenchymal origin showing histological, molecular, and clinical structures similar to their human equivalent. In human medicine, cellular myelocytomatosis (c-Myc) has already been proposed as a vital gene that regulates aggressive cell growth and is a predictive marker and possible therapeutic target in fibrosarcomas. The goal of this study was to evaluate c-Myc expression in canine and feline fibrosarcomas by gene and protein analyses and correlate its expression to the histological grading system of the examined cancers. Nineteen archival formalin-fixed and paraffin-embedded canine and feline sarcoma samples were recruited. Sixteen of them were determined to be canine and feline fibrosarcomas. The samples consisted of eight cutaneous samples, four eyelid samples, three oral mucosa samples, and one vulva sample. Histopathological examinations were performed to estimate histological type, differentiation, cellularity, and mitotic count. On each sample, immunohistochemical assessment and mRNA expression were performed using an anti-c-Myc antibody and a c-Myc primer. The differences in the immunoreactivity staining values and the relative folds of the c-Myc mRNAs were categorized using already recognized grading criteria and a statistical analysis was performed. Among all the tested canine and feline fibrosarcomas, 75% of the grade II fibrosarcoma cases and all of the grade III fibrosarcoma cases were positive for the c-Myc protein. Furthermore, the relative values of the c-Myc gene were up-regulated in grade III compare to grade I and II fibrosarcomas. The results demonstrated that c-Myc expression was significantly higher in grade III than in grade II and I (p ≤ 0.05) fibrosarcomas regarding protein and gene levels. In summary, this work provides evidence for the high expression of c-Myc in canine and feline fibrosarcomas and proposes a supposed relationship with poor prognoses as determined by grading systems.

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1. Introduction

Cancers are common in dogs and cats, with approximately one in every ten cats or dogs experiencing cancer during their lifetime [1]. Fibrosarcomas are highly malignant mesenchymal cancers derived from fibroblasts and connective tissue cells and are commonly observed on skin and subcutaneous tissue [2]. Even though fibrosarcomas happen in all domestic animals, they are more frequently seen in the skin of old cats and dogs [3]. In dogs, skin fibrosarcomas form more than 15% of all cancers [3,4]. The fundamental causes of fibrosarcomas are obscure; it has been suggested that changes in chromosomes 11 and 30, subcutaneous injections, trauma, or inflammation may contribute to cancer development in dogs [3,4,5]. In cats, skin fibrosarcomas are more common than in dogs [2,3]. They are usually seen after routine vaccination with rabies and feline leukemia virus vaccines [6,7].

Fibrosarcomas are constantly aggressive and fast-growing and can invade locally and through fascial planes and metastasize to other locations [2,8]. The metastatic foci are usually in the liver, lung, brain, and bones [9]. In spite of improvements in the field of veterinary medicine, dog and cat skin fibrosarcomas remain diagnostically challenging. Quick detection gives the best therapeutic consequences [10]. There is incomplete knowledge of the genetic processes underpinning soft tissue fibrosarcoma progression in dogs and cats. In human fibrosarcoma, a crucial research area is resolving the epigenetic and genetic causes that play an important role to these cancers, for example, the Myc gene [11]. The Myc proto-oncogenes (l-Myc, n-Myc, and c-Myc,) direct more than 15% of the transcription of expressed genes [12]. Myc’s key downstream mediators influence a huge variety of biological actions, for example, cell proliferation, differentiation, and apoptosis [11,12,13]. In mammals, c-Myc modulates several biological activities that are implicated in the cancer cell proliferation. Dysregulation of c-Myc is implicated in many abnormal conditions, for example, increased cell proliferation, genomic instability, immortalization, metastasis, and angiogenesis [12,14,15].

Normally, c-Myc is situated on chromosome F2 and 13 of cats and dogs, respectively [13]. In normal cells, c-Myc expression is very low because of a lack of a positive signaling pathway [14,15,16]. c-Myc expression has been considered to play a role in many cancers, for example, colorectal cancer [17], breast cancer [18], prostate cancer [19], and cervical cancer [20]. In humans, studies have found that c-Myc is highly expressed in many sarcomas including osteosarcoma, leiomyosarcoma, chondrosarcoma, rhabdomyosarcoma, and liposarcoma and it has been related to aggressiveness and a poor prognosis [20,21,22,23,24]. In human fibrosarcoma, c-Myc has revealed promise as a biomarker. Nevertheless, no reports have been issued concerning c-Myc association with dog and cat fibrosarcoma aggressiveness.

Overexpression of the c-Myc transcription factor is found in the nucleus of cancer cells and is connected to the family of basic helix–loop–helix–leucine zippers [25,26,27]. Using the relationship between immunohistochemistry and gene expression and histological grade of malignancy allows us to update the classification of cancer aggressiveness [28,29,30,31].

Therefore, this study aims to determine the expression of the c-Myc gene and protein in canine and feline soft tissue fibrosarcoma samples and the relationship between this marker and the histological grade of malignancy and to evaluate the probability of c-Myc being a valuable marker for this type of cancer.

2. Materials and Methods

2.1. Case Selection

The archives of the Pathology Department, College of Veterinary Medicine, University of Mosul were reviewed for cases of soft tissue carcinoma and sarcoma in canines and felines between 2012 and 2023. A total of 42 formalin-fixed and paraffin-embedded cancer tissues were included (27 canine and 15 feline samples). All of the cancer tissues were classified by two independent pathologists based on their diagnoses as carcinomas or sarcomas (Table 1). The soft cancers either originated from the skin, eyelid, oral mucosa, eyes, eyelids or vulva. A total of 16 soft tissue fibrosarcoma samples (9 canine and 7 feline) were nominated from among these cases.

2.2. Histopathological Examination

Histological sections (5 µm) from fibrosarcoma paraffin-embedded blocks were cut and stained with haematoxylin and eosin (HE). The canine and feline soft tissue fibrosarcoma samples were reassessed and further illustrated. Histologically, samples were further subclassified into a three-grade system [32,33,34]. According to cancer differentiation, cellularity, mitotic activity, and necrosis, the canine soft tissue fibrosarcomas were graded as grade I (low grade), grade II (intermediate grade) or grade III (high grade) [35]. In addition to these, in feline soft tissue fibrosarcoma, the number of inflammatory cells was considered an important part in the grading system [34]. The grading system was divided as follows: grade I, well-differentiated, low cellularity, low mitoses (0–9 mitoses/10 HPFs) and without necrosis and inflammatory cells; grade II, moderately differentiated and moderate cellularity, moderate mitoses (10–19 mitoses/10 HPFs) and with necrosis and inflammatory cells; grade III, poorly differentiated and high cellularity, high mitoses (more than 19 mitoses/10 HPFs), and high necrotic area and inflammatory cells. The slides were assessed and image analysis was performed using an Olympus microscope (Olympus, Tokyo, Japan) with a digital Omax image camera (Omax, Ningbo, China).

2.3. Immunohistochemical Staining

The slides were deparaffinized using xylene and rehydrated using a decreasing concentrations of alcohol; the peroxidase activity was removed by incubation for 15 min in 0.3% H_2_O_2_ in methanol. Then the slides were put in a citrate buffer of pH 6.00 and were microwave heated for 10 min. After washing in PBS, pH 7.4, the nonspecific proteins were blocked with 20% normal goat serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS. A polyclonal mouse anti-c-Myc primary antibody (Elabscience, Houston, TX, USA) dilution at 1:100 in PBS was placed overnight in a cold room. After washing with PBS, the slides were incubated in a rabbit anti-mouse secondary antibody (Elabscience, Houston, TX, USA) for 30 min diluted in 1:100 PBS. After washing, the sections were sensed with an avidin–biotin complex and then counter-stained with a hematoxylin solution for 1 min, washed, dehydrated, and cover-slipped. For the negative control, tissues were similarly preserved except that normal non-immune serum replaced the c-Myc antibody. Sections of human breast cancer (invasive ductal carcinoma) were used as positive control tissue [36]. The slides were observed using an Olympus microscope (Japan) with a digital Omax image camera (Omax, China). Ten fields (40×) from each cases were blindly assessed by two pathologists and each photo was expounded with (ImageJ Fiji/2.14.0, USA) to quantify c-Myc expression.

2.4. Immunohistochemical Assessment

C-Myc immunoreactivity was determined by a scoring system dependent on the staining intensity and positive cell percentage. The intensity staining was estimated as follows: (0) no staining; (1) weak staining; (2) moderate staining; (3) strong staining [37,38]. The positive cell score was evaluated as follows: negative (0); 1–25% immunopositive cells (1); 26–50% immunopositive cells (2); 51–75% immunopositive cells (3); ≥76–100% immunopositive cells (4) [37,39]. The end product was measured as the multiplication of the positive cell score and the staining intensity score.

2.5. Quantitative RT-PCR

In accordance with the histopathology results found, nine canine soft tissue fibrosarcomas (two from grade I, three from grade II, and four from grade III) and seven feline soft tissue fibrosarcomas (two from grade I, two from grade II, and three from grade III) were used for quantitation of the c-Myc gene. The total RNAs were collected from paraffin-embedded tissues using the FFPE RNA Kit (AmoyDx, Xiamen, China). The primers used for the c-Myc gene in the canine fibrosarcomas were as follows: forward primer, 5′-CCGTAACTCAAGATCGCCCC; reverse primer, 5′-CGCTCCACATGCAGTCCT and in the feline fibrosarcomas they were as follows: forward primer, 5′-TGGCTTGAAGGACACTGTTG; reverse primer, 5′-TGTTTCAACTGTTCTCGCCG, [40]. The integrity and quality of the mRNA was assessed using Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). Using reverse transcriptase, the mRNAs were transformed into cDNA. The RT-PCR mixtures for c-Myc and β actin were constructed with SYBR Green master mix, cDNAs, forward primer, reverse primer, and nuclease water. The mixture was put in a real-time PCR thermocycler machine. The differences in the relative folds of the c-Myc mRNAs between the grade I, II, and III soft tissue fibrosarcomas were measured in relation to the negative control (skin) and positive control (human breast adenocarcinoma).

2.6. Statistical Analysis

Statistical analysis was performed using the GraphPad software/10.6.1. The c-Myc immunoreactivity data were evaluated using the Mann–Whitney U test to estimate the differences between the grade I, II, and III soft tissue fibrosarcoma groups. A high significance was attributed when p < 0.001, while significance was attributed when p < 0.05. The relative mRNA values in the grade I, II, and III soft tissue fibrosarcoma samples were calculated as the mean of the three groups. The data were analyzed using a one-way ANOVA test in Microsoft Excel. The statistical significance level was set at a p value of <0.05.

3. Results

3.1. Analytical Details for Cancer Cases

An overall of 42 soft tissue malignant cases (27 canine and 15 feline) were found in the archives of the Department of Pathology, College of Veterinary Medicine. From all canine and feline malignant cases, carcinoma represented 55% (23/42) and sarcoma represented 45% (19/42). The histological types of fibrosarcoma percentage was 84% (16/19) from all sarcoma types. The ratio of canine fibrosarcomas to feline fibrosarcomas was approximately the same. At diagnosis, the mean ages of the dogs and cats were 12.5  ±  3.3 years and 10.4  ±  3.1 years, respectively. The canine and feline soft tissue fibrosarcomas most often affected the skin, in approximately 50% of all cases. The skin fibrosarcomas happened at the sites usually used for injections or vaccinations.

3.2. Histopathological Analysis of Fibrosarcoma

The histopathological results of the assessment consistent with the considerations stated earlier are summarized in (Table 2). A total of 16 samples diagnosed as canine and feline fibrosarcoma were investigated. The canine fibrosarcomas have been categorized into three grades: two were grade I (22%), three were grade II (33%), and four were grade III (45%). Grade I fibrosarcoma showed well-differentiated cancer cells within high collagen stroma, low cellularity, and minor atypia. Mitoses were rarely detected. Necrosis was not obvious (Figure 1a,b). Whereas, in grade II fibrosarcoma, cellularity and cellular atypia were increased with low interstitial collagen and some areas of necrosis (Figure 1c,d). However, the grade III fibrosarcoma had an abundance of cellularity, reduced collagen production, numerous atypical mitoses, high necrosis, and immature blood vessels (Figure 1e,f). The feline fibrosarcomas were categorized as grade I in 28.5% of cases, as grade II in 28.5% of cases and as grade III in 43% of cases. Four (47%) of the feline fibrosarcomas in this study displayed some or many spindle-shaped cells with some or many collagen fibers, an absence or low amount of mitoses, negative to moderate necrosis, and negative to mild lymphocyte infiltration and were graded as grade I or grade II (Figure 2a–d). Three (43%) of the feline fibrosarcomas in this study displayed high cellularity with cellular atypia, a low number of collagen fibers, a high number of mitoses, a large area of necrosis, and high perivascular lymphocytic infiltration and were diagnosed as grade III fibrosarcomas (Figure 2e,f).

3.3. c-Myc Immunoreactivity Evaluation

c-Myc expression was mainly detected in the nucleus of monomorphic spindle-shaped cancer fibroblasts (Figure 3). In canine and feline fibrosarcoma, c-Myc expression in grade I was positive in less than 5% of the cells (Figure 3a,b). However, in grade II canine and feline fibrosarcoma, the positive expression of c-Myc was observed in 87% and 56% of cancer fibroblasts, respectively (Figure 3c,d). Among all of the grade III canine and feline fibrosarcomas, approximately 100% of the cancer cells were positive for c-Myc (Figure 3e,f). The mean intensity staining c-Myc expression values were 25 ± 20.81 and 147.2 ± 57.26 for grade I and grade II canine and feline fibrosarcomas, respectively. The statistical evaluation exhibited a very significant difference between c-Myc expression for the grade I and grade II samples (p < 0.001) (Figure 4). The mean c-Myc expression values for the grade II and grade III canine and feline fibrosarcomas were 147.2 ± 57.26 and 283.33 ± 59.64, respectively. The statistical evaluation revealed a significantly higher expression (p ≤ 0.05) between these two groups (Figure 4). The statistical evaluation showed no significant difference between the grade III fibrosarcomas and the positive control (human breast cancer) in terms of their c-Myc expression.

3.4. Evaluation of c-Myc mRNA Expression

The relative values of the c-Myc mRNAs of 16 different canine and feline fibrosarcoma samples are exposed in Figure 5a. All fibrosarcoma samples exhibited abnormal relative values of c-Myc mRNA compared to normal samples, especially in grades II and III. The relative values of the c-Myc gene were irregularly up-regulated in grade III compare to grade I and II fibrosarcoma, and no clear variance in mRNA values was recognized when grade III fibrosarcoma was compared to breast cancer (positive control). Using a one-way ANOVA test, there is a highly significant correlation between the c-Myc mRNA values and the grade of fibrosarcoma with a p-value of <0.05 (Figure 5b).

4. Discussion

C-Myc has been one of the most common and significant oncogenes in cancer biology since its contribution to many vital processes was discovered [41]. It activates the expression of an unusual number of genes implicated in essential functions, for example, cell proliferation, DNA repair, the cell cycle and apoptosis [42,43]. c-Myc is highly expressed in almost all human cancers, certainly its overregulation leads to a numerous genetic variations, for example, chromosomal translocations and gene amplifications, which are linked with the abnormal proliferation of cancer cells [42,44]. c-Myc stimulates tumorigenicity through increased cell proliferation, permitting cancer cells to duplicate without control, stimulating genes participating in protein synthesis, or the encouragement of glucose consumption [45]. In addition, its overexpression generates cells that stimulate angiogenic factor production, for example, vascular endothelial growth factor, which motivates new blood vessel formation [46]. All these things make c-Myc a vital part of cancer progression, causative not only to cancer growth but also to the worsening of metastases and poor outcomes. In human oncology, the c-Myc oncogene shows an important part in the progression and development of many kinds of sarcoma [47]. In veterinary medicine, little is known about the degree of expression of c-Myc in canine and feline fibrosarcoma and its correlation with the malignancy grade of fibrosarcoma. In our study, we aimed to examine the c-Myc gene and protein expression in canine and feline fibrosarcoma archival samples which were categorized by a histopathological grading system into grades I, II and III.

In the retrospective assessment, dog and cat fibrosarcomas were the most common type of sarcoma cases and of these, the most commonly diagnosed were cancers of the skin. These results verify the findings of others [32,48,49]. In addition, fibrosarcomas were detected in older dogs and cats, which is coherent with previous studies defining the mean age at diagnosis of fibrosarcoma [50]. Actually, fibrosarcomas are malignant tumors, as normally believed. Many reports describe fibrosarcomas as low-grade fibrous masses that appear slow-growing [51,52]. In the current study, most (78% and 72%) of the observed canine and feline fibrosarcomas were grade II or III tumors and a strong relationship was detected between low differentiation, high cellularity, and high mitotic activity and the grade of fibrosarcoma.

The c-Myc protein is a nuclear marker linked to unrestrained cellular proliferation and it accelerates cell cycle passage and decreases several cell cycle checkpoints [24,53]. c-Myc expression take place in cells that are replicating, except in (G0) quiescent cells, forming a suitable marker for evaluating the tumorigenicity rate in many types of cancer [24,53,54]. In this research, we observed that c-Myc immunoreactivity is detected mostly in the nucleus of monomorphic spindle-shaped cancer fibroblasts. A high expression score was found in the grade II and III canine and feline fibrosarcoma samples. An even higher c-Myc expression score was noticed among grade II (75%) and III (100%) canine and feline fibrosarcoma cases in comparison with grade I (5%) cases. These results are in line with human medicine reports that confirmed that c-Myc is usually related to higher histological grades in different cancers and is frequently a marker of poor prognosis [55,56]. Regarding grading of cancer, we detected a significant strong relationship between c-Myc expression and cancer grade. Amongst grade I and grade II canine and feline fibrosarcoma cases, we showed a highly significant difference in c-Myc expression (p ≤ 0.001), while all samples of grade III canine and feline fibrosarcoma indicated high c-Myc expression that was the same as the positive control (human breast cancer) [18,27,39,56]. Our results were consistent with human medicine outcomes [54,57], therefore demonstrating a strong relationship between c-Myc expression and the degree of malignancy as imitated by the grading of canine and feline fibrosarcomas. Since grade is the best parameter generally used to display the stage of fibrosarcoma, the increased level of the c-Myc protein is significantly connected to c-Myc expression as a suitable prognostic marker for determining grade.

To corroborate this, further evaluation was arranged concerning the c-Myc mRNA levels of the canine and feline fibrosarcoma samples to evaluate whether c-Myc mRNAs were differentially expressed between grade I, II, and grade III samples. Our finding exposed that the relative fold values were elevated in all grade III fibrosarcoma samples compared to the levels in the grade I and II samples. Previously, expression of c-Myc in mRNA levels has been stated to be linked to poor prognosis in high-grade human solid cancers such as osteosarcoma, synovial sarcoma, liposarcoma, chondrosarcoma, and leiomyosarcoma [21,58,59]. For this reason, the status of c-Myc offers further prognostic indication in canine and feline fibrosarcoma, commonly representing a high-risk tumor, and may be a possible target for developing therapeutic plans, not only in veterinary medicine but also in human medicine.

As far as we know, this is the first study to examine the role of c-Myc expression in gene and protein levels and correlate it with the severity degree grading system for canine and feline fibrosarcoma. The limitations of this research comprise the small cohort size and the inadequate follow-up time, emphasizing the demand for the assessment of this biomarker in potential multicenter and worldwide examinations

5. Conclusions

These results prove that higher expression of the c-Myc gene and protein can be significantly linked to higher grades of canine and feline soft tissue fibrosarcoma, proposing its possible association to cancer development and progression. Nevertheless, since this paper proves associations instead of the causation of malignancy, additional pathway studies are essential to comprehend and interpret large lists of genes associated to the c-Myc gene. In addition, due to a lack of longitudinal clinical outcomes and clinical follow-up analysis, the current conclusions may not have prognostic significance and should be confirmed in future potential studies.