Corrigendum: Neutrophil to lymphocyte ratio and principal component analysis offer prognostic advantage for dogs with mammary tumors.

[This corrects the article DOI: 10.3389/fvets.2023.1187271.].

Neutrophil to lymphocyte ratio and principal component analysis offer prognostic advantage for dogs with mammary tumors.

Introduction: In veterinary medicine, cancer is the leading cause of death in companion animals, and mammary gland tumors represent the most common neoplasm in female dogs. Several epidemiological risk factors, such as age, breed, hormones, diet, and obesity have been reported to be relevant for canine mammary tumors. Nowadays, the gold standard for diagnosis of canine mammary tumors is the pathological examination of the suspected tissue. However, tumor grade can only be assessed after surgical removal or biopsy of the altered tissue. Therefore, in cases of tumors that could be surgically removed, it would be very helpful to be able to predict the biological behavior of the tumor, before performing any surgery. Since, inflammation constitutes part of the tumor microenvironment and it influences each step of tumorigenesis, cellular and biochemical blood markers of systemic inflammation, such as the neutrophil to lymphocyte ratio (NLR) and the albumin to globulin ratio (AGR) have been proposed as prognostic factors for human cancer development. The NLR and the AGR have not been explored enough as prognostic factors for cancer development in veterinary medicine. Methods: To determine the prognostic value of NLR in canine mammary tumors, clinical records including biochemistry and hematological studies of female dogs with mammary tumors and of control healthy dogs, were used to determine the pre-treatment NLR and AGR. Other clinical data included age, breed, tumor size, histological tumor grade, and survival time after surgery. Results and discussion: It was found that a higher pre-treatment NLR value (NLR > 5) associates with less survival rate. In contrast, the AGR did not show any predictive value on the malignancy of the tumor. However, by combining the NLR with AGR, age of the dog, and tumor size in a principal component analysis (PCA), the grade of the tumor and survival after surgery could be appropriately predicted. These data strongly suggest that pre-treatment NLR values have a prognostic value for the survival rate after surgery of dogs with mammary tumors.

The extracellular matrix protein pattern in the canine neoplastic mammary gland.

Extracellular matrix (ECM) proteins in the mammary gland provide structure and regulate its development and homeostasis. Alterations in its structure can regulate and support pathogenesis, like breast tumors. Aiming to identify the health and tumoral canine mammary ECM scaffold protein profile by immunohistochemistry, the decellularization process was carried out to remove the cellular content. Additionally, it was verified the influence of health and tumoral ECM on the attachment of health and tumoral cells. The types I, III, IV, and V structural collagens were scarce in the mammary tumor, and ECM fibers were disorganized. Vimentin and CD44 were more common in mammary tumor stroma, suggesting a role in cell migration that results in tumor progression. Elastin, fibronectin, laminin, vitronectin, and osteopontin were similarly detected under healthy and tumor conditions, providing the attachment of normal cells in healthy ECM, while tumoral cells were able to attach in tumoral ECM. The protein pattern demonstrates ECM alteration in canine mammary tumorigenesis, presenting new knowledge on mammary tumor ECM microenvironment.

Epidemiological, clinical, and histopathological aspects of mammary neoplasms in dogs from Rio Branco, Acre, Brazil / Aspectos epidemiológicos, clínicos e histopatológicos das neoplasias mamárias em cães do município Rio Branco, Acre, Brasil

Mammary neoplasms are very common in the clinical routine of small animals and have an enormous histological diversity and complexity. Most of these neoplasms are classified as malignant and originate from metastases, considered the main cause of death. This study aimed to present and correlate epidemiological data with the malignancy of histological types of mammary neoplasms in dogs in the municipality of Rio Branco, Acre, Brazil. The analyzed samples were collected in collaborating hospitals and veterinary clinics, the data acquired through a questionnaire filled in by them, the clinical staging carried out through the TNM system, and the histological classification according to the Consensus regarding the Diagnosis, Prognosis, and Treatment of Canine and Feline Mammary Tumors. The fixed material was evaluated macroscopically and then subjected to classical histological processing. Histological preparations were observed under optical microscopy. The data were organized statistically using Pearson's chi-square test (p<0.05). Of the 71 samples obtained from dogs, 50 consisted of mammary neoplasms, 18 skin neoplasms, and three non-tumor lesions. Eleven breeds of dogs were identified. Among them, 48 were females and two males with an average age of 10 years and most patients intact (90%) and the minority used progestogens (16%). Of the mammary neoplasms, 94% had a malignant profile, with carcinoma in a mixed tumor being the most frequent (60%). Statistically, no correlation was observed between breed, sex, age, reproductive status, use of progestogens, mammary location, or tumor size with malignancy. In general, dogs can be affected by malignant mammary neoplasms, regardless of the mentioned variables, reinforcing the importance of clinical staging and early diagnosis for the best prognosis within the small animal clinic.

As neoplasias mamárias são muito frequentes na rotina clínica de pequenos animais e possuem uma enorme diversidade e complexidade histológica. A maior parte dessas neoplasias é classificada como malignas e originam às metástases, sendo consideradas a principal causa mortis. Este estudo objetivou apresentar e correlacionar os dados epidemiológicos com a malignidade dos tipos histológicos das neoplasias mamárias em cães dentro do município de Rio Branco, Acre. As amostras analisadas foram coletadas em hospitais e clínicas veterinárias colaboradoras, os dados adquiridos através de questionário preenchido pelos mesmos, o estadiamento clínico realizado através do sistema TNM e a classificação histológica segundo o Consenso de Diagnóstico, Prognóstico e Tratamento de Neoplasias Mamárias Caninas. O material fixado foi avaliado macroscopicamente e, em seguida, submetido ao processamento histológico clássico. As preparações histológicas foram observadas sob microscopia óptica. Os dados obtidos foram organizados na forma estatística utilizando o Teste de Qui-quadrado de Pearson (p<0,05). Das 71 amostras obtidas em cães, 50 foram neoplasias mamárias, 18 foram tumores de pele e três eram lesões não tumorais. Foram identificadas 11 raças caninas, 48 eram fêmeas e dois machos, com idade média de 10 anos, onde a maioria dos pacientes eram inteiros (90%) e a minoria fazia uso de progestágenos (16%). Das neoplasias mamárias, 94% apresentaram o perfil maligno, sendo o carcinoma em tumor misto o mais frequente (60%). Estatisticamente não houve correlação entre raça, sexo, idade, estado reprodutivo, uso de progestágenos, localização das mamas ou tamanho tumoral com a malignidade. A espécie canina de modo geral pode ser acometida de neoplasia mamária maligna, independente das variáveis citadas, reforçando a importância do estadiamento clínico e diagnóstico precoce para o melhor prognóstico dentro da clínica de pequenos animais.

DNA Methylation Status of the Estrogen Receptor α Gene in Canine Mammary Tumors.

Estrogen receptor α (ERα) has an important role in mammary carcinogenesis, prognosis, and treatment. In human and canine mammary cancer, the most aggressive tumors show loss of ERα expression, which in human breast cancer has been attributed to methylation of the cytosine followed by guanine (CpG) island within the estrogen receptor α gene ( ESR1) promoter. This study aimed to investigate the role of ESR1 CpG island (CGI) methylation in ERα expression in canine mammary tumors. Twenty-one canine mammary samples were sorted into three groups: malignant tumor (n = 9), benign tumor (n = 8), and normal gland (n = 4). Immunohistochemical analysis and reverse-transcription quantitative real-time PCR were performed to assess ERα expression and ESR1 mRNA levels. The methylation status was determined using sodium-bisulfite-treated DNA sequencing. All normal mammary glands and benign tumors showed high ERα expression (score range, 5-8). Six of the nine malignant tumors did not show ERα expression (score 0), two had score 2, and one had score 4. Lower ERα ( P < .005) and ESR1 mRNA levels ( P < .005) were found in malignant mammary tumors than in the other two groups. Canine ESR1 has an intragenic and non-promoter-associated CGI, different from humans. No significant variation in methylation percentage was observed among the groups, suggesting that ESR1 is not regulated by DNA methylation, unlike that in humans. This difference should be considered in further research using ERα as a biomarker for mammary tumors in canine studies on ERα-targeting therapy.

Microambiente tumoral nas neoplasias mamárias em cadelas Revisão de literatura / Tumor microenvironment in canine mammary tumors - Literature review

Nos últimos anos, o microambiente tumoral foi associado à evolução de doenças crônicas, incluindo as neoplasias mamárias. Um crescente conjunto de evidências foi compilado para demonstrar o papel importante deste processo envolvendo as neoplasias mamárias caninas. A maneira pela qual as interações entre o tumor e o estroma participam ativamente do desenvolvimento neoplásico deve ser elucidada. Apesar de desempenhar função de contribuição na oncogênese, ainda não sabemos se este é um efeito direto ou indireto. A seguinte revisão descreve e esclarece algumas das principais evidências para a interação com o estroma tumoral e a evolução da progressão neoplásica.(AU)

In the recent years, the tumor microenvironment has been associated with the evolution of chronic diseases, including the mammary tumors. The evidence was compiled to demonstrate the important role of this process involving the canine mammary tumors. The way that the interactions between the tumor and the stroma actively participate in tumors development must be elucidated. Despite playing a role of contribution in the oncogenesis, we still do not know if this is a direct or indirect effect. The following review describes and clarifies some of the key evidence for the role of the interaction with the tumor stroma and the evolution of neoplastic progression.(AU)

Microambiente tumoral nas neoplasias mamárias em cadelas – Revisão de literatura / Tumor microenvironment in canine mammary tumors - Literature review

Nos últimos anos, o microambiente tumoral foi associado à evolução de doenças crônicas, incluindo as neoplasias mamárias. Um crescente conjunto de evidências foi compilado para demonstrar o papel importante deste processo envolvendo as neoplasias mamárias caninas. A maneira pela qual as interações entre o tumor e o estroma participam ativamente do desenvolvimento neoplásico deve ser elucidada. Apesar de desempenhar função de contribuição na oncogênese, ainda não sabemos se este é um efeito direto ou indireto. A seguinte revisão descreve e esclarece algumas das principais evidências para a interação com o estroma tumoral e a evolução da progressão neoplásica.

In the recent years, the tumor microenvironment has been associated with the evolution of chronic diseases, including the mammary tumors. The evidence was compiled to demonstrate the important role of this process involving the canine mammary tumors. The way that the interactions between the tumor and the stroma actively participate in tumor’s development must be elucidated. Despite playing a role of contribution in the oncogenesis, we still do not know if this is a direct or indirect effect. The following review describes and clarifies some of the key evidence for the role of the interaction with the tumor stroma and the evolution of neoplastic progression.

Envolvimento da matriz extracelular no tumor mamário canino / Extracellular matrix involviment in canine mammary tumor

A glândula mamária (GM) é um tecido dinâmico, derivado da epiderme e o seu desenvolvimento depende da interação entre as células mamárias e o estroma. A matriz extracelular (MEC) representa o principal conteúdo extracelular, responsável pela sustentação do tecido conjuntivo, da membrana basal e serve como reservatório para muitos fatores de crescimento. MEC é constituída por fibras proteicas insolúveis, como colágenos, lamininas, fibronectinas, e polímeros solúveis, como proteoglicanos e glicosaminoglicanos. Essas moléculas que compõem a MEC são importantes, tanto durante a morfogênese da GM, como para a sua manutenção conferindo-lhe a sustentação e o armazenamento de substratos necessários para seu crescimento. A desorganização da MEC na GM pode ser um indício necessário para o início e a progressão dotumor de mama. O Tumor mamário canino (TMC) é referido como um complexo de neoplasias que tem a participação de diversos fatores para seu desenvolvimento, incluindo os componentes da MEC. Desta forma, a investigação da MEC no diagnóstico dos TMC torna-se importante, para estabelecer a correlação entre os seus componentes e as células neoplásicas, além de fornecer informações sobre o comportamento biológico e o estadiamento clínico dos TMC. O entendimento da participação dessas moléculas da MEC para o desenvolvimento do TMC pode favorecer abordagens terapêuticas mais específicas, tendo como alvo elementos da MEC. Portanto, esta revisão tem como foco a participação dos componentes da MEC nos processos que contribuem para o estabelecimento do TMC, o que pode favorecer abordagens terapêuticas que visem elementos da MEC.(AU)

Mammary gland (MG) is a dynamic tissue derived from the epidermis and your development depends on the interaction between mammary cells and stroma. Extracellular matrix (ECM) is the major extracellular content of tissues responsible for supporting connective tissue and basement membrane, and serves as a reservoir for many growth factors. ECM is comprised of insoluble protein fibers as collagens, laminins, fibronectins and soluble polymers as proteoglycans, and glycosaminoglycans. The ECM components are important both during morphogenesis of MG as to maintain this fabric giving support and storage of substrates needed for the growth. ECM disorder in GM may be the progression trigger of the breast tumor. Canine mammary tumor (CMT) is a complex of malignancies that have the participation of several factors for its development, including ECM components. Therefore an investigation of ECM in the diagnosis of CMT becomes important to establish a relationship between componentes of matrix and neoplastic cells, including information on the biological behavior and clinical staging of CMT. The knowledge of ECM molecules participation in the development of CMT may further therapeutic approaches targeting elements of ECM. Thus, this review has a focus on the ECM components participation in the processes that contribute to CMT establishment, which may favor therapeutic approaches targeting elements of ECM.(AU)

Envolvimento da matriz extracelular no tumor mamário canino / Extracellular matrix involviment in canine mammary tumor

A glândula mamária (GM) é um tecido dinâmico, derivado da epiderme e o seu desenvolvimento depende da interação entre as células mamárias e o estroma. A matriz extracelular (MEC) representa o principal conteúdo extracelular, responsável pela sustentação do tecido conjuntivo, da membrana basal e serve como reservatório para muitos fatores de crescimento. MEC é constituída por fibras proteicas insolúveis, como colágenos, lamininas, fibronectinas, e polímeros solúveis, como proteoglicanos e glicosaminoglicanos. Essas moléculas que compõem a MEC são importantes, tanto durante a morfogênese da GM, como para a sua manutenção conferindo-lhe a sustentação e o armazenamento de substratos necessários para seu crescimento. A desorganização da MEC na GM pode ser um indício necessário para o início e a progressão dotumor de mama. O Tumor mamário canino (TMC) é referido como um complexo de neoplasias que tem a participação de diversos fatores para seu desenvolvimento, incluindo os componentes da MEC. Desta forma, a investigação da MEC no diagnóstico dos TMC torna-se importante, para estabelecer a correlação entre os seus componentes e as células neoplásicas, além de fornecer informações sobre o comportamento biológico e o estadiamento clínico dos TMC. O entendimento da participação dessas moléculas da MEC para o desenvolvimento do TMC pode favorecer abordagens terapêuticas mais específicas, tendo como alvo elementos da MEC. Portanto, esta revisão tem como foco a participação dos componentes da MEC nos processos que contribuem para o estabelecimento do TMC, o que pode favorecer abordagens terapêuticas que visem elementos da MEC.

Mammary gland (MG) is a dynamic tissue derived from the epidermis and your development depends on the interaction between mammary cells and stroma. Extracellular matrix (ECM) is the major extracellular content of tissues responsible for supporting connective tissue and basement membrane, and serves as a reservoir for many growth factors. ECM is comprised of insoluble protein fibers as collagens, laminins, fibronectins and soluble polymers as proteoglycans, and glycosaminoglycans. The ECM components are important both during morphogenesis of MG as to maintain this fabric giving support and storage of substrates needed for the growth. ECM disorder in GM may be the progression trigger of the breast tumor. Canine mammary tumor (CMT) is a complex of malignancies that have the participation of several factors for its development, including ECM components. Therefore an investigation of ECM in the diagnosis of CMT becomes important to establish a relationship between componentes of matrix and neoplastic cells, including information on the biological behavior and clinical staging of CMT. The knowledge of ECM molecules participation in the development of CMT may further therapeutic approaches targeting elements of ECM. Thus, this review has a focus on the ECM components participation in the processes that contribute to CMT establishment, which may favor therapeutic approaches targeting elements of ECM.

Downregulation of ATM Gene and Protein Expression in Canine Mammary Tumors.

The ataxia telangiectasia mutated (ATM) gene encodes a protein associated with DNA damage repair and maintenance of genomic integrity. In women, ATM transcript and protein downregulation have been reported in sporadic breast carcinomas, and the absence of ATM protein expression has been associated with poor prognosis. The aim of this study was to evaluate ATM gene and protein expression in canine mammary tumors and their association with clinical outcome. ATM gene and protein expression was evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in normal mammary gland samples (n = 10), benign mammary tumors (n = 11), nonmetastatic mammary carcinomas (n = 19), and metastatic mammary carcinomas (n = 11). Lower ATM transcript levels were detected in benign mammary tumors and carcinomas compared with normal mammary glands (P = .011). Similarly, lower ATM protein expression was observed in benign tumors (P = .0003), nonmetastatic mammary carcinomas (P < .0001), and the primary sites of metastatic carcinomas (P < .0001) compared with normal mammary glands. No significant differences in ATM gene or protein levels were detected among benign tumors and nonmetastatic and metastatic mammary carcinomas (P > .05). The levels of ATM gene or protein expression were not significantly associated with clinical and pathological features or with survival. Similar to human breast cancer, the data in this study suggest that ATM gene and protein downregulation is involved in canine mammary gland tumorigenesis.

The maspin expression in canine mammary tumors: an immunohistochemical and molecular study / A expressão do maspin nos tumores mamários caninos: um estudo imuno-histoquímico e molecular

The serpin maspin, a tumor suppressor in breast cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumor metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other types of cancer. Little is known about expression, regulation and function of maspin in canine mammary tumors. It was demonstrated in this study, a loss of maspin expression in malignant canine mammary cells compared with a pool of normal canine mammary tissue, analyzed by quantitative real-time PCR; weak maspin expression in malignant canine mammary tumors were observed by immunohistochemistry. It was also demonstrated that a correlation with nuclear maspin expression and a good prognosis. It is suggested that maspin could be used as a prognostic marker in canine mammary neoplasia.

O serpin maspin, um supressor tumoral no câncer de mama foi descrito como inibidor de migração celular e indutor de adesão celular entre a membrana basal e a matriz extracelular resultando na inibição da metástase tumoral. Por outro lado, a alta expressão do maspin está relacionada com um mau prognóstico em outros tipos de câncer. Pouco se sabe sobre a expressão, regulação e função do maspin nos tumores mamários caninos. Neste estudo, foi demonstrada uma perda da expressão de maspin nas células mamárias malignas de cães quando comparadas com um pool de tecido mamário normal de cães, analisado por PCR quantitativa em tempo real. Houve uma expressão fraca maspin em preparações de tumores mamários malignos observadas por imuno-histoquímica. Também foi verificado que a expressão nuclear do maspin em tumores mamários caninos está relacionada a um bom prognóstico. Assim, o maspin pode ser utilizado como um marcador prognóstico nas neoplasias mamárias em cães.