NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the “typical” form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation.Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in  TNNT1 and other forms with unusual presentation ..5].All patients who underwent muscle biopsy presented rods in muscle fibers. The distribution of rods inside muscle fibers was predominantly a mixed pattern of large subsarcolemmal rods associated with small intermyofibrillar rods .Among the 30 patients, 20 individuals  were followed over months to years (six months to ten years) in a neuromuscular outpatient clinic by multidisciplinary teams.Serial cardiac evaluations performed in these 20 patients over a period of up to ten years did not indicate any cardiac abnormalities.Three patients died with respiratory complications .NEB variants were present in 20 patients (P9–P28) from 16 families . Five families showed heterozygous mutations in the ACTA1 gene (P1–P5). In addition, in four families, mutations in the following genes were found: TPM2 (P6), TPM3 (P7/P8) and KLHL40   .NEB mutations, 17 individuals were classified as typical  and 3 with the mild form (childhood/juvenile form) .Among the 20 patients (16 families) with NEB variations, including family 16 and 23 with two siblings. Parental segregation analysis performed in three cases confirmed the presence of the two mutations in trans .All families showed two nebulin mutations, including point mutations, small deletions or duplication and CNVs (P9–P28). In two families, there were two affected siblings (P11/P12 and P23/P24) carrying homozygous mutations, and family 10 , we found a mutation in only one allele of the  Chip v3  confirmeNEB gene, ten were in the splice sites, nine were frameshift, three stop-gain, one non-frameshift duplication and two CNVs. Eleven of these variants were newly described mutations: four frameshift , three splice site , one stop-gain (c.16423A>T), one non-frameshift duplication (c.6869_6870insTGC) and two CNVs .Among the total of twenty-five different identified variants in the Four variants were recurrent: c.24579G>C in three families , c.22170G>A; p.Tyr7390* in two families (P14 and P17), c.24189_24192dup; p.Glu8065Serfs*5 in two families ; and c.5343+5G>A in two families (P23/P24 in homozygous and P27/P28 in heterozygous).Although mutation c.24579G>C was detected in three patients from different families, they were from the same state of Brazil. Among these three patients, two  showed the same and previously described mutations in compound heterozygosity (c.22170C>A and c.24579G>C).NEB mutations could be performed in 11 individuals with the typical form . Among these patients, three died suddenly because of respiratory complications . The age of death for P10, P20 and P26, was, respectively, 25 years, 15 years and 5 years of age.The follow-up of patients with Although three patients  were classified as the typical form, they showed more intense weakness and needed ventilatory support and gastrotomy in the first two years of life. P10 presented with repetitive pneumonia and, at 18 months of age, he had tracheostomy and needed mechanical ventilation 24 h/day, and he died at 25 years of age due to respiratory complications. P21 had tracheostomy at the age of 8 months, during a respiratory infection. At the age of 5 years, the tracheostomy could be replaced by non-invasive ventilation (NIV). At her last clinical evaluation, she was 12 years old, still using gastrostomy and NIV for more than 16 h/day. P25 started using NIV at 7 months of age and is still using it for more than 16 h/day. He developed a progressive weakness, losing the capacity to control the head and to sit and needed a gastrostomy at 5 years old. These three patients did not achieve the capacity to walk, and all presented severe scoliosis.Four patients  were using NIV, which was started during puberty. Two patients (P14 and P17) were using it during nighttime, and two patients were using it for more than 16 h each day (P15 and P16). All these patients were able to walk. However, one lost this ability at 17 years old (P16). In the first years of life, P16 showed proximal weakness in the upper and lower limbs, compatible with the typical form. During puberty, she developed an intense and progressive involvement of distal muscles in the lower limbs, which impaired her capacity to walk during adolescence. The weakness progression was accompanied by respiratory failure and severe scoliosis. At the age of 15, she was admitted for spinal surgery but had persistent severe respiratory failure, needing NIV almost 24 h a day.P23 and P24, with the mild form of NM, are siblings and have shown a mild proximal and facial weakness with a stable course until now and are able to walk. They did not have ventilatory insufficiency or scoliosis at the last evaluation, at the age of 27 and 25 years, respectively.ACTA1 gene, four with the typical  and one with the severe form (P3).Five patients had mutations in the ACTA1 gene, and four of them were previously described missense variants: c.541G>C, p.Asp181His; c.478G>A, p.Gly160Ser; c.593G>A, p.Arg198His; and c.854T>G, p. Met285Arg.All five presented heterozygous mutations in the In P2, we found a novel splice site intronic mutation (c.130-5T>A), classified as a variant of uncertain significance (VUS), following the ACMG pathogenicity classification guidelines, but it showed some pathogenic indicatives based on in silico prediction programs, including the Human Splicing Finder and MaxEntScan. This mutation was not present in the father or the mother and was considered a de novo mutation.ACTA1 mutations was performed in four patients, three with the typical  and one with the severe form (P3).The follow-up of patients with Two patients  with the typical form did not need ventilatory support and were able to walk until the last evaluation. They did not present scoliosis; however, P5 was followed until the beginning of her puberty, and P2 had his last evaluation at 13 years old. One patient (P1) walked at 5 years of age but started using a wheelchair before 11 years of age and needed NIV in adult life.P3 presented severe weakness since birth. He uses ventilatory support 24 h a day with tracheostomy and never acquired the capacity to walk. He presented an asymmetric weakness distribution. He showed a more intense weakness on the left side of the body and on the right side of the face. At the age of 13 years, he needed spinal surgery to correct a severe scoliosis.TPM2 gene and presented the typical form. He developed mild scoliosis during puberty and had been followed without the necessity for surgical procedure. He does not need ventilatory support. The clinical and genetic data were previously described [One patient, P6, showed the recurrent mutation p.K7del in the escribed .TPM3 gene. Both patients presented with the typical form. P7 was 19 years old at the last evaluation; he developed a progressive scoliosis during puberty and needed NIV ventilation during nighttime. At the age of 15 years, he had scoliosis surgery. His father, P8, is 62 years old and started using NIV at the age of 51. Both have stable motor function and are still able to walk.Two patients from the same family (P7 and P8) showed a previously described mutation  in the KLHL40 gene. In patient 29, a segregation  study in the parents revealed that the previously described p.Gly469Ser mutation was present in the father [Two patients  showed two identical heterozygous mutations in the Patient 30 also presented with the severe form and showed severe hypotonia and weakness in the face and limb muscles associated with multiple arthrogryposis since birth. She started using NIV at two years after multiple pneumonias. She was able to sit at two years of age. At the age of 5 years, she is still not able to stand up or walk independently, and she has scoliosis. In this patient, according to the laboratory report (Invitae), the mutations were also present in different alleles.The slides from the muscle biopsy of each patient were reviewed, and the data are provided in In 28 patients, muscle biopsy was performed and showed rods inside the muscle fibers. Type I predominance was present in all patients, and in some, there was total predominance.In two patients (P24 and P29), muscle biopsy was not performed; P24 had a sibling who had already had a muscle biopsy, and in P29, it was not necessary because the molecular diagnosis was conclusive.Muscle imaging of the lower limbs was performed in eight patients  and whole-body MRI in one patient (P6) .Among the patients with nebulin mutations, muscle imaging was performed in five cases . In P16, muscle tomography was performed because she could not tolerate the supine position for a long time. This patient showed a diffuse and moderate involvement of thigh muscles and accentuated fat replacement in the legs .In case 21 with a severe weakness, we observed moderate involvement of the thigh muscles  and leg muscles  . In the ACTA1 mutations were submitted for muscle MRI. P3, with a severe clinical form, presented accentuated and diffuse involvement in the thigh and the leg. In P2, with the typical form, we observed a mild involvement of the anterior tibialis muscle with no abnormalities in the thigh.Two patients (P2 and P3) with TPM2 mutation, the whole-body MRI showed facial involvement, mainly temporal and lateral pterygoid muscles, and distal involvement in the lower legs, mainly soleus and flexor digitorum muscles  involved in NM.Over the last 20 years, we have been evaluating Brazilian NM patients, characterizing their clinical phenotype and morphological and molecular aspects ,46,47. IACTA1 genes [According to several studies still using the old clinical classification, the predominant NM forms are the typical, followed by the intermediate form, and the severe and childhood/juvenile forms ,3,27. InA1 genes ,44,48.During the follow-up of 20 NM patients, we observed the importance of monitoring the respiratory function, since all patients with severe forms, as well as a proportion of patients with the typical form, needed ventilatory support. Among the twenty patients with the typical form, three died because of respiratory complications, and six needed NIV despite preserving appendicular muscle strength and the ability to walk. This pattern of respiratory involvement out of proportion with the appendicular skeletal muscles is common in NM, which can mask the risk of respiratory complications in the follow-up of these patients ,49,50. TThe histologic hallmark of NM is the presence of nemaline bodies or rods inside the muscle fiber . The preNEB and de novo dominant mutations in ACTA1 [ACTA1 mutations (20%), two families with KLHL40 mutations (8%) and one family with mutations in TPM2 (4%) and TPM3 (4%). Therefore, we were able to genetically classify all 25 families, which is a great advance, achieved mainly because of the possibility to use more powerful molecular tools.Genetically, NM is very heterogeneous, including more than 12 genes to date . The mosin ACTA1 ,35. ConsConsidering the clinical manifestations, our findings are in accordance with the literature, since the most common clinical form related to nebulin mutations in our patients was the typical form, observed in 17/20 of our patients, followed by the mild childhood form 3/20) [/20 3,5,,5,27.NEB, but in some cases, it can be insufficient, and it would be necessary to combine additional techniques, such as CGH microarray and RNA analysis [NEB gene (TRI) and is frequently difficult to analyze in exome sequencing. In 2016, Kiiski et al. [NEB TRI variation in 13% of the families and in 6% of the controls. They identified that the NEB gene tolerates deviation of one TRI copy, but two or more might be pathogenic. So, in our patient P21, we suggest that the CNV could be pathogenic. The exon 29 deletion (P22) was observed using the software XHNM but not when the Motor Chip v3 was used. This last mutation needs to be better studied.The molecular screening of variants in the nebulin gene is very challenging, since this gene contains 183 exons, and in the middle of the gene, there is a triplicated region (TRI) consisting of a perfectly repetitive series of 8 exons , and there are three regions with alternatively spliced exons  ,53. NGS analysis . In the i et al.  used theNEB patients, at least one truncating mutation was found .The prevalence of different types of mutations in nebulin was estimated as follows: splice site mutations (34%), frameshift mutations (32%), small (<20 bp) deletions or insertions, nonsense mutations (23%) and missense mutations (7%). Large deletions and duplications (>1 Kb) were considered rare (4%) . SimilarAdditionally, we identified eleven novel variants in Brazilian patients: eight were truncating, two CNVs and one non-frameshift duplication (VUS). In four patients, a novel truncating variant was associated with another previously described variant , and in two siblings, a novel truncating variant was found in homozygosity (P11/P12). So, in these five families, the genetic defect could probably lead to protein truncation or degradation. Among the identified CNVs, the duplication of exons 82–105 (P21) was associated with a previously described truncating mutation, and the segregation study showed this is a “de novo CNV”. The novel 29 exon deletion (P22) was also associated with a novel truncating mutation, and both were classified as pathogenic. In patient 13, the non-frameshift duplication, classified as VUS, was in association with a previously described truncating variant. However, in these last two patients, we could not perform a segregation study, and we need more studies to confirm the pathogenicity and consequence of mutation associations.NEB: one frameshift mutation in exon 168 associated with a splice site mutation in exon 182, which could damage the SH3 domain. Similarly, a patient with a homozygous mutation in exon 185, which showed an absence of labeling with the SH3 antibody, was also described in 1999 [NEB–SH3 mouse model, generated by knocking in a nonsense codon in the last exon, eliminating the C-terminal SH3 domain of nebulin, no nebulin expression abnormalities or altered localization of the interaction partners of the SH3 domain were observed [P20 was previously described in 2002, when we observed a lack of the C-terminal domain of the nebulin protein (antibody SH3) through Western Blot analysis in the muscle tissue of this patient . In the observed . The mouobserved . It is iThe most frequent mutation described in different populations is the p.Arg2478_Asp2512del deletion encompassing exon 55 (del55)  and in tACTA1 gene encodes the skeletal muscle alpha-actin protein, which is the predominant actin isoform in the sarcomeric thin filament. Mutations in ACTA1 are the second most frequent cause of nemaline myopathy. More than 177 mutations were described in the ACTA1 gene, including missense, nonsense, frameshift and splice site mutations. Most mutations are dominant and de novo; only 10% of the pathogenic variations are recessive. In our cohort, we identified five families with ACTA1 heterozygous mutations in sporadic cases. Four patients were clinically classified with the typical form and one with the severe form. Considering the type of mutations, four of them were missense mutations previously described in the literature [The terature ,35.One novel intronic splice site variant (intron 2) was found in P2 and was classified as a VUS. A variant in the same nucleotide (c.130-5T>C) was classified as benign due to the high frequency in two databases . However, the variant identified in our patient (c.130-5T>A) was analyzed using in silico prediction software, such as Human Splicing finder and MaxEntScan, and was also absent in the parents. This might indicate that this variant has a pathogenic effect but requires additional studies to fully prove its pathogenicity.ACTA1 located in a cluster between amino acid residues 139 and 165 [A severe clinical phenotype with the presence of intranuclear rods has been associated with variants in  and 165 . P3 in tACTA1 gene have been described by Lonarge et al., 2020 [Our P3 presented an asymmetric weakness distribution, with a more intense weakness on the left side of the body and on the right side of the face. The cause of this asymmetry is not clear. Interestingly, two patients with asymmetric weakness due to a mosaic pattern of mutations in the l., 2020 . HoweverTPM3, TPM2, TNNT1, KBTBD13, CFL2 (COFILIN2), KLHL40, KLHL41, LMOD3, MYO18B, MYPN, RYR3, TTN3, ADSSL1, Filamin C and MYH2 [TPM3 gene showed an autosomal dominant inheritance, with a father and a son presenting a proximal lower limb weakness. The son presented a severe progressive scoliosis requiring spine surgery. Our TPM2-related patient is a sporadic case, carrying the recurrent mutation p. K7del, presenting the typical NM form with limitation of the opening of the mouth [Other genes have been related to NM in a small percentage of patients, including and MYH2 ,14,39. The mouth .KLHL40. This autosomal recessive nemaline myopathy (NEM8) was first described in 2013 in 20% of the patients with the very severe neonatal form, including fetal akinesia or hypokinesia, contractures, fractures and respiratory and swallowing difficulties apparent at birth. The average age at death was 5 months [Two of our patients with the severe NM form were compound heterozygous for the same two missense mutations  in KLHL40 in two unrelated families, since it is a very rare condition. The families are from the same state in Brazil (Sao Paulo), which is very large and has an ethnically heterogeneous population. However, we could not exclude a possible founder effect of these mutations or unknown relatedness between these two families.Additionally, an interesting point is finding the same genotype in Magnetic resonance imaging has been considered a powerful method for the study of muscle diseases. In nemaline myopathy, some patterns of muscle involvement are related to the mutated gene; however, a complete correlation between the gene and muscle commitment is not recognized yet .In the present study, among the patients with nebulin mutations, we observed a pattern of selective muscle involvement related to the clinical severity of the disease, independently of age. In patients with the typical form (mild phenotype), the thigh muscles were spared, and the anterior tibial and soleus muscles were more affected in the leg. In patients with a more severe weakness, the thigh muscles were also affected, including the vastus lateralis, rectus femoris, hamstring muscles, and in the leg, mainly the anterior tibial and soleus muscles were involved, similar to previously described findings . In one ACTA1 mutations and the mild phenotype showed diffuse involvement in the thigh, mainly in the adductor magnus and vastus muscles, and in the legs, the tibial anterior and the soleus were more affected. We could perform muscle MRI in two patients with mutations in ACTA1, one with a typical form and mild weakness and the other with a severe form and accentuated weakness. In both patients, we observed that the pattern of fat infiltration in muscle MRI was related to the severity of the clinical phenotype.In the literature, muscle MRI in patients with TPM2 mutation, using whole-body MRI, we could confirm the classical pattern of muscle involvement, with the predominant alterations in masticatory and distal leg muscles [Considering the patient with a  muscles .TPM3-related myopathy are scarce. In 2014, Schreckenbach et al. [TPM3. The muscle MRI showed a more intense involvement of the posterior compartment in the thigh and in the legs; the more affected muscles were the tibial anterior and soleus. Differently, in our patient, the thigh muscles were mildly affected, more intensely in the sartorius and adductor magnus muscles, while in the legs, the anterior compartment was more involved.Data on muscle imaging in h et al.  publishePatients with NM were evaluated over the last 20 years in two reference centers for NMD in Sao Paulo and Minas Gerais. Additional patients were referred from several other states of the country.The study included a total of 30 patients from 25 apparently unrelated Brazilian families. Patients were clinically evaluated, and some of them were followed for months to years by the same pediatric neurologist and multidisciplinary team. Based on clinical disease severity, the patients were classified into six groups: severe, intermediate and typical congenital forms, a mild childhood/juvenile-onset form, adult-onset NM and atypical forms.Muscle biopsy was performed for diagnostic purposes. Cryostat sections of 8 μm were submitted to conventional histological and histochemical techniques: hematoxylin and eosin (H&E), modified Gomori trichrome (GO), periodic acid Schiff technique (PAS), Oil Red O (ORO), reduced nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase (NADH-TR), succinic dehydrogenase (SDH), cytochrome c oxidase (COX) and adenosine triphosphatase (ATPase) pre-incubated at pH 9.4, 4.63 and 4.35.The presence of rods was analyzed in the GO stain and multiminicores or cores on NADH reaction. Type I predominance was considered when the proportion of type I fibers was more than 55% of the total fibers in quadriceps and more than 60% in deltoid muscles ,48. TherGenomic DNA was extracted from blood using standard protocols.http://www.abraom.ib.usp.br/, accessed on 22 August 2022). Rare variants, mainly in the ACTA1, TPM2 and TPM3 genes, were selected and analyzed using bioinformatics tools. Gene mutation databases HGMD, LOVD and ClinVar were consulted in search of previously described pathogenic variants. The American College of Medical Genetics and Genomics (ACMG) pathogenicity classification guidelines [Next-generation sequencing was performed using a customized NGS panel with 95 genes involved in neuromuscular disorders (NMD) or whole-exome sequencing using Illumina’s Nextera kits for library preparation and custom capture of these genes or Agilent 44 Mv2 SureSelect Exon enrichment kit. Sequencing was performed on an Illumina MiSeq sequencer or Hiseq 2000. After filtering, the variants were compared to the control populations of the 1000-genome (NIH) and 6500-exome sequencing project from the Washington University and the recently created Online Archive of Brazilian Mutations—AbraOM   and through Motor Chip v3, a genomic hybridization microarray for copy number mutations in 245 genes related to neuromuscular disorders developed by Vincenzo Nigro’s Lab .ACTA1 (NM_001100), TPM2 (NM_003289.4), TPM3 (NM_152263.4), KLHL40 (NM 152393.4).Mutations are reported according to the cDNA reference sequences of nebulin (NM_001164507.1), We confirmed the inheritance in four families by Sanger sequencing samples from the parents.Muscle imaging was performed in patients able to collaborate without sedation. The imaging protocol was performed following the standard sequences: Axial T1-weighted (turbo/fast) spin echo, Axial T2-weighted (turbo/fast) spin echo with fat suppression. The slice thickness was 5–8 mm with interslice gap of 1–2 mm and in-plane resolution of 1–2 mm. We studied the lower extremities or the whole body.Muscle tomography of the lower limb was performed in patients who could not tolerate the recumbent position.For the image analysis, we used the semi-quantitative visual rating scale described by Mercuri et al. in 2002 and Wattjes et al. in 2010 ,43.NEB gene were the most common cause of NM, followed by mutations in the ACTA1 gene, in this cohort of Brazilian patients. In total, 34 different variants were identified in this cohort, 12 of them being newly described mutations. The NEB mutation c.24579 G>C was recurrent in three unrelated patients, but from the same state of Brazil, with a high frequency of consanguinity, suggesting a common ancestor. Two patients with severe forms of the disease presented the same KLHL40 mutations in compound heterozygosity, although their families were not related.The molecular analysis in the present study showed that mutations in the The most frequent form of NM was the “typical form”, which can be caused by mutations in different genes, and phenotypic variability can be observed among patients with mutations in the same gene. Respiratory involvement was very common in NM patients and could be out of proportion with limb weakness. This aspect is very important and needs special attention in the follow-up of these patients.Muscle MRI could show different patterns of muscle involvement associated with the affected gene, which can be useful in highlighting this gene for a more detailed analysis during the NGS screening for mutations. On the other hand, considering the same gene, this pattern can be variable with the severity of clinical presentation.NEB mutations and the complexity of this gene, NGS tools should be combined with techniques designed to detect CNVs, especially in patients with only one identified mutation.Considering the high frequency of


Human gastric cancer is a prevalent cancer worldwide with a high mortality rate. Although sharing many other features, the incidence of gastric cancer is lower in dogs than in humans. Surgery is the first-line treatment; however, it is associated with several complications. Nevertheless, chemotherapy to treat canine gastric cancer has not received much attention, probably due to its late diagnosis, fast progression, low median survival time, and very high mortality rate, along with the lack of publications with concrete scientific results. In this review, we explore the pharmacological approach used in treatment of this often-fatal disease. Human gastric cancer (GC) ranks as the fifth most prevalent cancer worldwide, and is the third leading cause of cancer-related death. The incidence of GC is lower in dogs than in humans, accounting for less than 1% of all canine malignancies. In recent years, efforts have been made to understand the pathogenesis of GC and in find an appropriate therapy to maximize curative results, such as adjuvant chemotherapy treatments in addition to surgery. Although surgery is the first-line treatment, it is associated with several complications. In terms of chemotherapeutic intervention, canine gastric cancer has not received much attention, probably due to its late diagnosis, fast progression, low median survival time, and very high mortality rate, along with the lack of publications with concrete scientific results. In this review, we explore canine GC and the pharmacological approach used in the treatment of this often-fatal disease. Human gastric cancer (GC) ranks as the fifth most prevalent cancer worldwide, and is the third most common cause of cancer-related death . The incIn recent years, progress has been made in understanding the pathogenesis of GC, and efforts have been carried out to find appropriate therapies that could maximize curative results, such as adjuvant chemotherapy treatments in addition to surgical removal alone . In human medicine, there are factors that influence treatment, especially when the tumor is advanced, recurrent, metastatic, or even inoperable. In those cases, in order to promote a high median survival time and provide lifetime quality, chemotherapy is highly recommended as an adjuvant therapy  or neoadjuvant therapy  ,10. DepeIn veterinary literature, limited information is available regarding the clinical outcome of GC after surgical resection, as well as chemotherapy’s role in the progression of this disease. Indeed, studies evaluating dogs that have had surgery to treat gastric carcinoma have been restricted to case reports and case series that included multiple gastric tumor types . In this review, canine GC and the different approaches used in the treatment of this disease, often fatal or associated with a poor prognosis, are thoroughly described. Helicobacter pylori infection playing an important role [The etiology of human GC is complex and not completely understood, with hereditary cancer syndromes, known risk factors, other gastric diseases, and ant role . In dogsant role . Severalant role ,7,13,14,ant role ,7,14,15,ant role ,16,17, aant role .As in humans, clinical signs of GC in dogs are usually mild to absent during the early stages of the disease, with the most common being vomiting, anorexia, weight loss, and lethargy ,19,20. TAbout 90–95% of GC in humans are adenocarcinomas, which originate from the epithelial cells of the gastric mucosa. Similarly, adenocarcinoma is also considered the most common neoplastic entity in the stomach of dogs, comprising 50–90% of all canine gastric malignancies ,4,21.A preliminary diagnosis is usually obtained using ultrasound or endoscopic examination of the stomach, allowing the mucosa visualization and the collection of biopsies in order to enable a definite diagnosis . Due to Microscopically, according with the WHO classification for domestic animals , carcinoThe prognosis of GC cases is poor, and 70–90% have metastasized by the time of diagnosis or euthanasia ,6,7,14. Reported survival times in untreated dogs are less than 3 months after the onset of clinical signs . Surgical treatment of gastric neoplasia consists of pylorectomy and gastroduodenostomy (Billroth I) ,7,24, alSeveral studies described putative complications associated with the surgery aiming to treat canine GC and their association with the overall animal survival . ThirteeA study assessed perioperative (intraoperative and/or postoperative) complications in dogs that underwent surgical resection for gastric adenocarcinoma. Ten out of the total number of patients were subjected to partial gastrectomy (≤70% of the stomach removed). Of these: (a) two dogs displayed major intraoperative and postoperative complications, such as spillage of gastric contents and septic peritonitis, surviving only 1 day; and the other had an inadvertent stab incision into the small bowel and septic peritonitis, surviving 190 days; (b) three patients had postoperative major complications, such as cardiopulmonary arrest (surviving 2 days); septic peritonitis (surviving 2 days); and gastric stasis, pancreatitis, and cardiopulmonary arrest (surviving 15 days); (c) three dogs presented intraoperative complications with minor hemorrhage, surviving 2, 3, and 177 days, respectively; (d) two dogs had minor postoperative complications—hypertension was verified in one animal that achieved 274 days of survival time, and the other exhibited hyporexia, surviving 132 days . FurtherFive out of the total patients underwent the Billroth I surgical procedure . Among these: (a) one patient had pancreatitis as a major postoperative complication, surviving 16 days; (b) another suffered hypertension as a minor intraoperative complication, as well as ascending cholangiohepatitis and pancreatitis as postoperative complications, and had a survival time of 71 days; (c) another animal presented minor intraoperative complications, such as a major hemorrhage and a 2nd-degree atrioventricular block, and had a survival time of 49 days; (d) the last two dogs exhibited major postoperative complications—one presented severe pancreatitis and intermittent hypoglycemia and survived 258 days, while the other pulled out the gastrostomy tube, but survived 183 days . Four paIn another two patients that were subjected to a subtotal gastrectomy : one displayed a major intraoperative complication that lead to a subtotal gastrectomy and postoperative complications such as septic peritonitis, surviving 13 days; the second patient presented a minor intraoperative hemorrhage and postoperative complications such as vomiting, accomplishing a survival time of 93 days . Another canine patient with gastric adenocarcinoma that suffered total gastrectomy was euthanized after 240 days due to discomfort exhibited during and after eating . Veterinary oncology is an expanding area, and chemotherapy protocols have been established in common canine neoplasms. The most common treatment of canine GC is wide surgical resection in order to obtain clean margins. However, due to nonspecific clinical signs, the disease is often identified in an advanced stage, and its extension and location usually precludes a surgical approach ,11,19,20Although still unclear, chemotherapy protocols used in various studies include single agents or combination therapies employed after surgical resection see  4,11,1212,26,27.In terms of chemotherapeutic intervention, canine GC has not received much attention, probably due to its rare occurrence, late diagnosis, fast progression, low median survival time, and very high mortality rate, along with a lack of publications with concrete scientific results.Platinum drugs are an important class of antitumor compounds. Alone or in combination with other agents, cis-diamminedichloroplatinum (II) (cisplatin) and its analogues display a significant impact on the treatment of various solid tumors. However, the high toxicity profile observed with cisplatin has led to the development of platinum analogues that are more tolerable and active against different tumor types, including some that are resistant to cisplatin . In addition to cisplatin, two other platinum complexes are currently approved for use: cis-diamminecyclobutanedicarboxylato platinum (II) (carboplatin) and 1,2-diaminocyclohexaneoxalato platinum (II)  . The ant2 of carboplatin is administered intravenously (IV) every 3–4 weeks, while 350 mg/m2 is administered in dogs larger than 15 kg [Carboplatin is a second-generation platinum-compound chemotherapeutic agent that can be safely used in companion animals. It was developed for humans in an attempt to minimize the numerous side effects of cisplatin, including nephrotoxicity, nausea, vomiting, neurotoxicity, ototoxicity, and myelosuppression, maintaining comparable tumor cytotoxicity ,31,32,33an 15 kg ,31.2 carboplatin for the first dose and 200 mg/m2 for the subsequent treatments, three times in a period of 10 weeks. Despite the fact that the lesion inspired a poor prognosis, the status of the patient was well controlled after treatment achieving 30 months of survival after surgery [2 IV every 3 weeks, being administered five of the six intended doses. The survival time after surgery was 272 days, with the animal presenting grade I lethargy, vomiting, diarrhea, and grade II neutropenia [2 IV every 3 weeks, and the survival times were 93 days with grade I neutropenia after the 3rd dose and 383 days with grade I diarrhea , respectively [Carboplatin alone was used in some studies aiming the treatment of canine GC, although different protocols were followed: (a) 250 mg/m surgery ; (b) 280tropenia ; (c) twoectively . 2+ homeostasis, inhibition of thioredoxin reductase, and interaction with plasma membrane components. DOX is administered intravenously and is extensively distributed to all tissues [Antitumor antibiotics are natural products that originate from microbial fermentation, including anthracyclines (doxorubicin) and a synthetic analogue of the anthracenediones (mitoxantrone), mitomycins, and actinomycins, which have produced clinically useful compounds with various mechanisms of action. Doxorubicin (DOX) reacts with a variety of cellular components, and its activities include DNA intercalation and inhibition of RNA and DNA polymerases and topoisomerase II, alkylation of DNA, reactive oxygen generation, perturbation of cellular Ca tissues ,29,30,312 after a formal cardiac evaluation [The dose-limiting toxicities associated with DOX therapy are infusion-rate-dependent hypersensitivity, myelosuppression, gastrointestinal toxicity, and a cumulative dose-related cardiotoxicity ,29,30,31aluation ,29,30,31aluation ,29,31. Daluation ,29,31. 2 every 3 weeks for dogs larger than 15 kg, and 1 mg/kg for smaller patients [In dogs, the conventional dosing administration is 30 mg/mpatients ,29,30,312 IV was administered, but with a different frequency for each. One dog began systemic chemotherapy with DOX on day 43 after surgery, and it was repeated on days 69, 90, and 111. The patient responded well to therapy initially; however, after day 90 (3rd DOX dose), the dog began to lose weight, and on day 114, it presented extreme lethargy, fever, increased respiratory effort, and recurrent pneumonia, and was euthanized [Doxorubicin alone was used in four cases of canine gastric adenocarcinoma; in all cases, 30 mg/mthanized . Anotherthanized . In anotthanized .Mitoxantrone is a synthetic DOX analogue that has the same activity of DOX in DNA intercalation and inhibition of RNA and DNA polymerases and topoisomerase II ,29,30,31This drug is extensively distributed to tissues, and minimal levels are long-lasting after IV administration. A portion of mitoxantrone is not metabolized <30%) and is excreted unchanged in the urine and feces ,10,29,31% and is 2 IV slow bolus every 3 weeks) is used as a cardiac-sparing anthracycline when the patient reaches the cumulative level of DOX or there is evidence of cardiomyopathy and the patient is at risk of damage with DOX administration [In dogs, mitoxantrone  was used in a dog with gastric adenocarcinoma as a palliative intracavitary chemotherapy to treat metastatic bicavitary effusion. It was well tolerated and provided a survival time of 311 days after surgery [Intracavitary mitoxantrone  is an analogue of difluorinated deoxycytidine that has an important clinical activity against numerous human solid tumors and hematologic malignancies ,29,31,39This drug is administered through an IV route, being relatively well tolerated when used as a single agent. The main dose-limiting toxicity consists of myelosuppression, but in the presence of other antimetabolites, gemcitabine produces greater hematologic toxicity in longer infusions ,39. In h2 IV over 20 to 30 min, every week for 4 weeks) or low-dose  options, depending on the use of other cytotoxics [In veterinary oncology, gemcitabine has been rarely reported, although some studies used it as a single agent or in drug combinations, or combined it with radiation treatment. In dogs, dose regimens involve both high-dose  is a halogenated analogue of uracil that enters cells via the facilitated uracil base transport system and is intracellularly converted to active nucleotide forms via phosphorylase and kinase reactions to yield monophosphate, diphosphate, and triphosphate forms of both fluorouridine and fluorodeoxyuridine, which are incorporated into RNA and DNA and interfere with its synthesis and function ,30,31,392 of the reference dose IV weekly [In dogs, 5-FU can be administered topically and intralesionally, and causes a dose-dependent myelosuppression, gastrointestinal toxicity, and neurotoxicity. However, 5-FU is rarely used for management of epithelial tumors , with the protocol for this being 150 mg/mV weekly ,29,30,31In veterinary medicine, there are two kinase inhibitors approved for use in dogs: masitinib and toceranib . MasitinToceranib is a tyrosine kinase inhibitor that has antiangiogenic and antitumor properties via the targeting of molecules such as platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), KIT protooncogene receptor tyrosine kinase (KIT), FMS-like tyrosine kinase 3 (FLT3), and colony-stimulating factor 1 receptor (CSF1R). Toceranib has demonstrated activity in mast cell tumors, sarcomas, and carcinomas, and has been used in the treatment of various solid tumors ,31,40.A study assessed the treatment with toceranib of different types of canine adenocarcinomas . The results suggested that the effects of toceranib might depend on the type of adenocarcinoma. The dogs tolerated the therapy well, although they presented a higher rate of adverse events when compared to those treated with surgery alone. Postoperative adjuvant therapy with toceranib was associated with a longer time to progression when compared to those treated with surgery alone .In another study, five dogs with gastric adenocarcinoma were treated with toceranib after surgery using different dose regimens and frequencies: (i) 1.7 mg/kg PO on Monday/Wednesday/Friday (MWF); (ii) two doses of 2.7 mg/kg PO MWF; (iii) 3 mg/kg PO on MWF; (iv) two doses of 1.5 mg/kg PO on MWF; and (v) 3.4 mg/kg PO on MWF. In three dogs, this treatment was finished shortly after initiation due to the adverse effects, such as grade II diarrhea and grade I lethargy; in the other two cases, toceranib was well tolerated and was continuously used until disease progression was noted. The survival time of this therapy ranged from 49 to 403 days  .Alkylating agents are a group of anticancer drugs whose mechanism of action consists of the covalent binding of alkyl groups to cellular macromolecules; these agents target DNA, in which they form mono- or bifunctional adducts that produce inter- or intrastrand cross-links ,30,45. Cyclophosphamide is a nitrogen mustard prodrug that involves cytochrome p450 metabolism to release active alkylating species ,31,45. CThe alkylating agents are frequently used as single- or multiagent protocols in the treatment of different types of cancer, with cyclophosphamide as the most widely used alkylating agent against a great variety of tumors . The primary dose-limiting toxicity of these agents is bone marrow suppression (neutropenia), with secondary restraining effects in the proliferation of intestinal mucosa cells ,30,31,452) by PO or IV route in the dog, or used as a metronomic chemotherapy, in which it is administered long-term in a continuous basis at low doses and with minimal pauses [In dogs, gastrointestinal toxicity is not common. However, sterile hemorrhagic cystitis and alopecia in breeds with continually growing hair are frequent manifestations of toxicities ,29,30,31Prednisone  and prednisolone  are synthetic analogues of cortisol. Prednisone is converted to prednisolone in the liver. Both can be used alone or in combination with other immunosuppressive drugs to treat transplant rejection and autoimmune diseases. Prednisolone is administered via IV as a prodrug ,46,47,482 PO daily [In companion animals, prednisolone is used in the treatment of lymphoid neoplasias, mast cell tumors, and brain tumors. This drug is well tolerated in dogs in a short time period. Usually the patients receive 2 mg/kg or 40mg/mPO daily ,31. PredPO daily . A patient with gastric adenocarcinoma that suffered mucosal resection of the pylorus was treated with 0.5–1.0 mg/kg/day of prednisolone, and the survival time recorded was at least 104 days without recurrence, as the animal was alive at the end of the study .Some drugs were not conceived to be used as an anticancer therapies, but some of them can inhibit angiogenesis, and therefore exhibit an antineoplastic profile; these include inhibitors of matrix metalloproteinase (MMP) and cyclooxygenase (COX). Piroxicam is a nonsteroidal anti-inflammatory drug that belongs to the oxicam family ,30,31,50The potential of piroxicam as a COX inhibitor in the treatment of angiogenesis has been studied in canine transitional cell carcinoma ,31,52. IA dog with gastric adenocarcinoma was treated with 0.3 mg/kg/day of piroxicam; however, the animal died 374 days later due to tumor progression . The effectiveness of the combination of different chemotherapy drugs when compared to single-agent therapies is due to the overcoming of the natural and acquired resistance of tumor cells and the use of agents that diverge in dose-limiting adverse effects. In humans, when compared to the single agent, combination chemotherapy has shown a curative potential in some malignancies. In veterinary oncology, combination chemotherapy was successful in the treatment of canine lymphoma .2 carboplatin IV was administered after surgery, followed by five doses of 200 mg/m2 carboplatin plus 150 mg/m2 5-FU IV every 3 weeks, without adverse effects and with a survival time of 553 days, as the was patient alive at the end of the study [2 5-FU was administered, and 1 h later, 200 mg/m2 IV of carboplatin was added over 10 min, and the survival times were 79 and 26 days, respectively [Some investigations used combination-chemotherapy protocols as a treatment approach to canine GC . Carboplhe study . Anotherhe study . In two ectively . 5-FU caectively . Neverthectively .2 IV carboplatin every 3 weeks, with grade III neutropenia, and the survival time registered was 354 days [2 of gemcitabine plus 285 mg/m2 of carboplatin IV on week 1, and four cycles of 57 mg/m2 IV of gemcitabine on week 2, with neutropenia and grade II thrombocytopenia, resulting in 564 days of survival time. The same circumstances occurred in a case in which six doses of 5 mg/m2 IV mitoxantrone were administered every 3 weeks, and after chemotherapy, one dose of 240 mg/m2 IV carboplatin, with a grade I elevation in creatinine. The survival time was 190 days [In one dog with gastric adenocarcinoma, toceranib administration (2.5 mg/kg on MWF for 3 months) began after cytotoxic chemotherapy of six doses of 285 mg/m354 days . In anot190 days .2 every 3 weeks), cyclophosphamide (2 months of 15 mg/m2 daily), and doxorubicin , and the survival time achieved was 274 days without side effects [A multiagent protocol to treat gastric adenocarcinoma used a combination of carboplatin . Two different protocols of doxorubicin and cyclophosphamide were administered in cases of adenocarcinoma: in the first, the patient received 27 mg/m2 IV of doxorubicin in week 1, while in weeks 2 and 3, the patient received 222 mg/m2 PO of cyclophosphamide divided over 2 days, fulfilling two of the five intended cycles. The animal reached 101 days of survival time without side effects reported [2 IV of doxorubicin and 50 mg/m2 PO of cyclophosphamide for 4 days after surgery due to the presence of metastasis. This patient was euthanized 9 weeks after diagnosis due to recurrent vomiting. When used in combination to treat canine gastric adenocarcinoma, 5-FU (150 mg/m2 IV)/cyclophosphamide (two cycles of 50 mg/m2 PO for 4 days every 2 weeks), the owner elected euthanasia 9 weeks after the diagnosis due to the progression of vomiting [2 IV), cyclophosphamide (75 mg/m2 PO for 4 days in week 1), and 5-FU (8 cycles of 150 mg/m2 IV in weeks 2 and 3), plus two cycles of 60 mg/m2 IV of cis-platinum over 6 h every 3 weeks. The patient died 7.5 months after diagnosis due to acute anuric renal failure [2/day). The survival time was 1366 days, and the cause of death was attributed to gallbladder rupture [Cyclophosphamide can be used in combination with different drugs, such as toceranib, doxorubicin, 5-FU, and piroxicam. In order to treat adenocarcinoma, 1.7 mg/kg PO of toceranib was applied on MWF and 2.5 mg/mreported ; the sec rupture .Another protocol consisted of endoscopic polypectomy and the administration of 0.3 mg/kg/day of piroxicam and 0.5–1.0 mg/kg/day of prednisolone. The survival time was 1250 days without recurrence, and the dog was alive at the end of the study .Nowadays, companion animals assume a major role in human families. The balance between the quality and duration of such animals’ lives can be a sensitive matter, but hardly understandable by the owner due to the stronger pet–owner relationship. Therefore, in recent years, an increasing demand has been observed in the search for basic and specialized treatment of animals with cancer ,56,57. TCancer management in companion animals should begin with a discussion with the pet owner about the potential risks, benefits, toxicity, costs, and time commitment of treatment . Pain enGenerally, surgery is the best treatment modality if the primary tumor can be completely removed with adequate morbidity. However, some tumors may be difficult to excise due to extensive involvement ,55. 2). In addition, breed-specific characteristics can induce side effects [Chemotherapy is used to reduce or prevent tumor spreading, but has a limited therapeutic index and can cause substantial or fatal toxicity, so the negative side effects and the beneficial outcome that this therapy can have on the prognosis and survival time in animal patients should be evaluated ,60. In v effects . A study investigated the factors that may influence owners to use chemotherapy to treat their animals. It suggested that owners’ beliefs, their previous experiences with chemotherapy, and their consciousness of adverse effects over possible benefits may influence their choices in using this treatment in their animals. A reduction in the quality of life was expected during and after the treatment by the majority of the participants. However, most of the owners thought there would be an improvement in the life quality of their animals after chemotherapy, but the survival time after therapy was overestimated, which led to false expectations. Nevertheless, fewer owners would choose this treatment if the extended survival time was less than 3 months, demonstrating that animals’ life quality is important in the decision process. Interestingly, the owners chose chemotherapy treatment based on their perspective, rather than that of their animals. Thus, it is imperative to enlighten owners regarding the chemotherapeutic procedure and its effects by emphasizing the life quality, incidence, and expression of the adverse effects, so they can make rational and informed choices .Survival time is frequently used as the primary end point because it is easy to determine. However, in veterinary oncology, it can be affected by owner-driven factors such as delaying the therapy’s initiation or euthanasia . SurvivaSome limitations of these studies included a small sample (due to the rare occurrence of canine GC), a lack of knowledge regarding the disease’s clinical stage, and variations in the surgical procedure protocol, as well as the fact that, in most of the cases in which treatment was attempted, postmortem examinations were not performed ,11,38. OIn humans, chemotherapy improves survival, and combination therapies also improve survival when compared to a single-agent treatment. In patients subjected to target therapy with HER-2-positive tumors, protocols using trastuzumab in combination with capecitabine or 5-FU in combination with cisplatin have proven to be valuable. For those patients with tumours that are HER-2 negative, protocols with two- and three-drug combinations, such as those including irinotecan, docetaxel, oxaliplatin, or oral 5-FU, are effective therapy options for advanced gastric cancer . With so few reported cases, most which involved a single patient, it is difficult to correctly evaluate the response to postoperative chemotherapy of canine GC. Thus, it is extremely important to perform preliminary and thorough studies using in vitro models. The testing of anticancer drugs using cancer cell lines presents other advantages than just cytotoxicity evaluation tests, because it permits researchers to analyze the actions of drugs and of combinations of them, as well as to screen for resistance/sensitivity. The results of research in cancer cell lines are usually extrapolated to or tested in in vivo tumors , and may