COX‐2, mPGES‐1 and EP2 receptor immunohistochemical expression in canine and feline malignant mammary tumours

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E₂ (PGE₂) tumour‐promoting activity has been confirmed and its production is controlled by Cyclooxygenase‐2 (COX‐2) and microsomal PGE synthase‐1 (mPGES‐1). Evidence suggests that mPGES‐1 and COX‐2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX‐2, mPGES‐1 and EP2 receptor expression in canine (n = 46) and feline (n = 50) mammary tumours and in mammary non‐neoplastic tissues. COX‐2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES‐1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX‐2, EP2 receptor and mPGES‐1 expression was significantly higher in carcinomas than in non‐neoplastic tissues and adenomas. COX‐2, mPGES‐1 and EP2 receptor expression was strongly associated. These findings support a role of the COX‐2/PGE2 pathway in the pathogenesis of these tumours.     

High COX‐2 expression is associated with increased angiogenesis,proliferation and tumoural inflammatory infiltrate in canine malignant mammary tumours: a multivariate survival study

COX‐2 expression affects mammary tumourigenesis by promoting angiogenesis and cell proliferation, encouraging metastatic spread and tumour‐associated inflammation. Samples of canine mammary tumours (n = 109) were submitted to immunohistochemistry to detect COX‐2, CD31, VEGF, Ki‐67, CD3 and MAC387 expression. Concurrent high expression of COX‐2/CD31, COX‐2/VEGF, COX‐2/Ki‐67, COX‐2/CD3 and COX‐2/MAC was associated with elevated grade of malignancy, presence of intravascular emboli and presence of lymph node metastasis. Tumours with high COX‐2 (P < 0.001) and tumours with concurrent expression of high COX‐2 and high CD31 (P = 0.008); high VEGF (P < 0.001); high Ki‐67 (P < 0.001); high CD3+ T‐lymphocytes (P = 0.002) and elevated MAC387 macrophages (P = 0.024) were associated with shorter overall survival (OS) time. Interestingly the groups with high COX‐2/CD31 and high COX‐2/VEGF retained their significance after multivariate analysis arising as independent predictors of OS. Present data highlight the importance of COX‐2 in canine mammary tumourigenesis.     

The selective cyclooxygenase‐2 inhibitor mavacoxib (Trocoxil) exerts anti‐tumour effects in vitro independent of cyclooxygenase‐2 expression levels

The inducible inflammatory enzyme cyclooxygenase‐2 (COX‐2) and its product prostaglandin E2 (PGE₂) are prominent tumour promoters, and expression of COX‐2 is elevated in a number of tumours of both humans and canines. Targeting COX‐2 in cancer is an attractive option because of readily available non‐steroidal anti‐inflammatory drugs (NSAIDs), and there is a clear epidemiological link between NSAID use and cancer risk. In this study, we aim to establish the anti‐tumourigenic effects of the selective, long‐acting COX‐2 inhibitor mavacoxib. We show here that mavacoxib is cytotoxic to a panel of human and canine osteosarcoma, mammary and bladder carcinoma cancer cell lines; that it can induce apoptosis and inhibit the migration of these cells. Interestingly, we establish that mavacoxib can exert these effects independently of elevated COX‐2 expression. This study highlights the potential novel use of mavacoxib as a cancer therapeutic, suggesting that mavacoxib may be an effective anti‐cancer agent independent of tumour COX‐2 expression.     

Evaluation of histological grade and histologically tumour‐free margins as predictors of local recurrence in completely excised canine mast cell tumours

Completeness of mast cell tumour (MCT) excision is determined by assessment of histologically tumour‐free margins (HTFM). The HTFM width necessary to prevent local recurrence (LR), recognized as histologic safety margin (HSM) in human oncology, has not been defined. We hypothesized that HTFM width would correlate with risk for LR and high‐grade tumours would require wider HTFM than low‐grade tumours. Records of dogs with completely excised MCTs were included. Signalment, two‐tier tumour grade, tumour size, HTFM width, recurrence and therapy data was collected. High‐grade (n = 39) tumours were more likely to recur than low‐grade (n = 51) tumours (35.9% versus 3.9%), P < 0.0001, with no association between HTFM width and LR. Twenty‐nine percent of low‐grade tumours had HTFM less than 3 mm; none recurred. Narrow (≤3 mm) histologic margins are likely adequate to prevent LR of low‐grade tumours. High‐grade tumours have significant risk of LR regardless of HTFM width.     

Retrospective evaluation of COX‐2 expression,histological and clinical factors as prognostic indicators in dogs with renal cell carcinomas undergoing nephrectomy

Limited veterinary literature is available regarding prognostic markers for canine renal cell carcinoma (CRCC). We retrospectively evaluated COX‐2 expression, histological and clinical features associated with prognosis of CRCC. Sixty‐four cases post‐nephrectomy were included, 54 had histopathological assessment and 30 had COX‐2 immunostaining performed. Eight dogs (13%) had metastatic disease at initial diagnosis. Twenty‐seven dogs (42%) received adjuvant therapy after nephrectomy. On univariate analysis, COX‐2 expression, mitotic index (MI), histologic type, vascular invasion, neoplastic invasiveness and metastasis at diagnosis were significantly associated with overall median survival time (MST). COX‐2 score (COX‐2 score > 3 MST 420 days versus 1176 days if COX‐2 score <3; P = 0.011) and MI (MI > 30 MST 120 days versus 540 days for MI < 30; P = 0.003) were the only variables associated with CRCC outcome on multivariate analysis. The addition of MI and COX‐2 immunostaining to standard histopathological evaluation would help predicting outcome in CRCC patients.     

The animal‐dependent risk factors in canine T‐cell lymphomas

Malignant lymphomas are one of the most common malignancies occurring in dogs; among them T‐cell tumours are less commonly recognized. Recently, many authors have recommended cytology as a sufficient diagnostic method for canine lymphomas, especially if supported by immunocytochemistry or flow cytometry. The aim of the study was to characterize animal‐dependent risk factors in canine T‐cell lymphomas (TCLs) in Poland, including specific cytological subtypes. Determination of the type and subtype of the tumour was made based on the updated Kiel cytological classification adopted for dogs as previously described. Two breeds turned out predisposed to TCL (dog de Bordeaux and Boxer) while no predisposition to B‐cell lymphoma could be evidenced. Dogs with low‐grade lymphoma were significantly older than those with high‐grade lymphoma.     

High COX‐2 expression in canine mast cell tumours is associated with proliferation,angiogenesis and decreased overall survival

COX‐2 overexpression is associated with several hallmarks of carcinogenesis such as proliferation, angiogenesis, invasion and metastasis. Fifty cases of canine mast cell tumours (MCT) were retrospectively evaluated and submitted to immunohistochemistry for COX‐2, CD31, Ki‐67, MAC‐387 and CD3. Furthermore its relationship with clinicopathological variables and overall survival (OS) was analysed. COX‐2 intensity (P = 0.016), but not COX‐2 extension nor score was associated with decreased OS and higher grades of malignancy according to Patnaik (P = 0.002) and Kiupel (P < 0.001) grading systems. Cox‐2 intensity was also associated with higher Ki‐67 scores (P = 0.009), higher mitotic index (P = 0.022) and higher microvascularization density (P = 0.045). No association was observed for COX‐2 intensity and CD3‐T lymphocyte (P = 0.377) and macrophage infiltration (P = 0.261) by MAC‐387 immunollabelling, suggesting an active role of COX‐2 in MCT oncogenesis mainly through proliferation and angiogenesis stimulation making it a potentially clinical relevant prognosis marker and therapeutic target.     

In situ c‐KIT mRNA quantification of canine cutaneous mast cell tumours and its relationship to prognostic factors

Cutaneous mast cell tumours (MCTs) are the most frequent malignant skin tumours in dogs. Mutations in the c‐KIT proto‐oncogene are correlated with the pathogenesis and aggressiveness of MCTs. To date, studies have focused on c‐KIT mutations and KIT protein localization, with a general lack of mRNA‐level analyses. In this study, c‐KIT mRNA expression was investigated in canine MCTs by RNA in situ hybridization (RNA‐ISH). Furthermore, we evaluated associations between c‐KIT mRNA expression and the histological grade, KIT immunohistochemical staining pattern and other clinicopathological parameters. c‐KIT mRNA expression was observed in all MCT samples, appearing as clusters of dots in the cytoplasm of neoplastic cells. A significant correlation was detected between c‐KIT mRNA expression (quantified according to the H‐score and the percentage of positive cells) and the histological grade (determined using two‐and three‐tier grading systems; P < .05). We also found a significant positive correlation (all P < .05) between c‐KIT mRNA expression and the proliferation indices (mitotic index, Ki‐67, and Ag67). However, no significant associations with c‐KIT expression from RNA‐ISH were found with respect to different KIT staining patterns. Overall, these results demonstrate that c‐KIT mRNA expression might be an additional tool for measuring the c‐KIT status in canine cutaneous MCTs and could serve as a potential prognostic factor. Further studies should evaluate the prognostic significance of c‐KIT mRNA expression in a large and uniform cohort of canine MCTs.     

Tumour necrosis factor‐related apoptosis‐inducing ligand induces apoptosis in canine hemangiosarcoma cells in vitro

Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is an apoptosis‐inducing cytokine that shows potential therapeutic value for human neoplasms, and is effective in some canine tumours; however, its potential for killing canine hemangiosarcoma (HSA) cells is unknown. Thus, we evaluated the proapoptotic effect of TRAIL in nine canine HSA cell lines. Cells (JuA1, JuB2, JuB2‐1, JuB4, Re11, Re12, Re21, Ud2 and Ud6) were cultured with three recombinant human TRAILs (rhTRAILs): TRAIL‐TEC derived from Escherichia coli, TRAIL‐TL derived from mammalian cells and isoleucine zipper recombinant human TRAIL (izTRAIL) containing an isoleucine‐zippered structure that facilitates trimerization. TRAIL‐TEC did not decrease the cell viability in any of the cell lines tested, whereas the other two rhTRAILs effectively decreased the viability of all cell lines as assessed by the WST‐1 assay. In canine HSA cells, izTRAIL induced apoptosis more effectively than TRAIL‐TL. In JuB4, Re12, and Ud6 cells, izTRAIL increased the activation of caspase‐3 and caspase‐8 and caused poly (ADP‐ribose) polymerase degradation. Moreover, izTRAIL treatment increased the proportion of Annexin V+/ Propidium iodide (PI)? apoptotic cells and nuclear fragmentation in izTRAIL‐sensitive cells. These results show that rhTRAIL can induce apoptosis in canine HSA cells, but the sensitivity of TRAIL was different depending on the cell lines. Therefore, TRAIL could be an effective therapeutic agent against canine HSA, but the specific mechanism of resistance should be determined to clarify under what conditions this treatment would be most effective.     

In vitro Inhibition of Canine Complement‐Mediated Hemolysis

Background

Immune‐mediated hemolytic anemia (IMHA) is the most common hematologic immune‐mediated disease in dogs. Complement fixation on erythrocytes causes hemolysis. Complement inhibition decreases hemolysis in people with the hemolytic disease and also may prove effective in treating IMHA in dogs.

Hypothesis/Objectives

Evaluate the in vitro efficacy of 2 complement inhibitors used in humans against canine complement.

Methods

The inhibitory activity of the C3‐inhibitor compstatin and recombinant human C1‐esterase inhibitor (C1‐INH) was evaluated using an in vitro hemolytic assay and spectrophotometric measurement of released hemoglobin. Dose‐response curves for each inhibitor were generated.

Results

Compstatin decreased approximately 50% of canine complement‐mediated hemolysis in initial experiments. This inhibition largely was lost when a new lot of drug was purchased. C1‐INH showed a dose‐dependent inhibition. The highest concentration of C1‐INH tested (500 μg/mL) decreased >80% of canine complement‐mediated hemolysis, and the lowest concentration tested (31.25 μg/mL) decreased hemolysis >60%.

Conclusions and Clinical Importance

Human C1‐INH is a robust inhibitor of canine complement‐mediated hemolysis, whereas compstatin was minimally and variably effective. Human C1‐INH may substantially decrease complement‐mediated hemolysis in dogs with IMHA and warrants further investigation.     

A complication probability study for a definitive‐intent,moderately hypofractionated image‐guided intensity‐modulated radiotherapy protocol for anal sac adenocarcinoma in dogs

Previous trials showed the importance of administering radiation therapy (RT) with small doses per fraction in canine pelvic tumours to maintain acceptable toxicity levels. With increased accuracy/precision of RT, namely intensity‐modulated RT (IMRT), this approach might be challenged. Theoretical toxicity calculations for a new definitive‐intent moderately hypofractionated RT protocol for canine anal sac adenocarcinomas (ASAC) were performed, focussing on the risk of toxicity in pelvic organs at risk (OAR). Computed tomography datasets of 18 dogs with stage 3b ASAC were included. Re‐planning with margins for daily image‐guidance/IMRT was performed and a new protocol isoeffective to previously described definitive‐intent protocols was computed. Dose‐volume information were derived from individual plans and used for normal tissue complication probability (NTCP) computations. A 12 × 3.8 Gy protocol was computed for risk estimation. Tumour volumes ranged from 27.9 to 820.4 cm³ (mean 221.3 cm³ ± 188.9). For late rectal toxicity/bleeding ≥grade 2, median risk probability was 2.3% inter quartile range (IQR: 5.9; 95% confidence interval (CI): 1.2, 8.4) (rho = 0.436) and 3.4% (IQR: 0.96; 95%CI: 3.1, 4.0) (rho = 0.565), respectively. Median late toxicities in urinary bladder, kidneys and small bowel were <1%, except in one kidney. Myelopathy/myelonecrosis had a median risk probability of 4.1% (IQR: 23.5; 95%CI: 2.1, 25.2) (rho = 0.366) and 5.6% (IQR: 13.5; 95%CI: 3.1, 14.1) (rho = 0.363), respectively. However, graded risk showed a probability estimate for late spinal cord toxicity of ≥5% in 8/18 patients. The daily‐imaging IMRT 12 × 3.8 Gy protocol for canine ASAC seems tolerable for most cases, even in advanced disease. Theoretical dose computations serve as estimate, but are safe measures before implementing new protocols into clinical use.     

Evaluation of a multi‐agent chemotherapy protocol combining dexamethasone,melphalan, actinomycin D,and cytarabine for the treatment of resistant canine non‐Hodgkin high‐grade lymphomas: a single centre's experience

The DMAC protocol (dexamethasone, melphalan, actinomycin‐D, cytarabine) has been evaluated in American studies for the treatment of relapsed canine lymphoma, comparing similarly to other rescue protocols. The aim of this study was to evaluate efficacy and toxicity of DMAC, in a larger UK cohort of resistant canine lymphomas. Medical records of dogs with resistant non‐Hodgkin high‐grade lymphomas that received DMAC as a rescue protocol were reviewed from 2007 to 2017. Response, time from initiation to discontinuation (TTD) and toxicity (Veterinary Cooperative Oncology Group criteria) were assessed. One hundred dogs were included; 86 received CEOP (modified CHOP including epirubicin) as first‐line treatment. Thirty‐five dogs (35%) responded: 21 complete responders (CRs) and 14 partial responders (PRs). Responders had significantly longer TTD (P < 0.001) compared with non‐responders: 62 days (range 28‐952) for CR vs 32 days (range 20‐70) for PR. Six CR received more than six cycles of DMAC (range 7‐36 cycles) and experienced a longer TTD (median 508, range 126‐952 days). Thrombocytopenia occurred in 45% (24 grade 1‐2, 21 grade 3‐4) and neutropenia in 36% of cases (29 grade 1‐2, 7 grade 3‐4). Gastrointestinal toxicity occurred in 42% of dogs (40 grade 1‐2, 2 grade 3‐4). Owing to chemotherapy toxicity, treatment was discontinued in five, and hospitalization required in six cases. In this study, response to DMAC was lower and of generally shorter duration than previously reported. Toxicity was high, but infrequently led to hospitalization or discontinuation of treatment.     

Increased expression of tissue inhibitor of metalloproteinase‐1 correlates with improved outcome in canine cutaneous mast cell tumours

Canine mast cell tumour (MCT) is a biologically heterogeneous disease. The extracellular matrix degradation promoted by matrix metalloproteinases (MMPs) has been studied in an attempt to elucidate the mechanisms involved in the biological behaviour of tumours. The aim of this study was to characterize the expression of MMP‐2 and ‐9 and tissue inhibitors of metalloproteinase (TIMP)‐1 and ‐2 in canine cutaneous MCTs and to evaluate their prognostic values. Immunohistochemical staining for MMP‐2, MMP‐9, TIMP‐2 and TIMP‐1 was performed in 46 canine cases of MCTs. TIMP‐1 expression showed an independent prognostic value for post‐surgical survival and disease‐related mortality. Dogs with MCTs showing less than 22.9% mast cell TIMP‐1 positivity were more prone to die because of the disease and had a shorter post‐surgical survival. This article suggests the involvement of TIMP‐1 in MCT progression, by contributing to a good outcome in patients with MCTs.     

Ketoprofen pharmacokinetics of R‐ and S‐isomers in juvenile loggerhead sea turtles (Caretta caretta) after single intravenous and single‐ and multidose intramuscular administration

Ketoprofen is a nonsteroidal anti‐inflammatory and analgesic agent that nonselectively inhibits cyclooxygenase, with both COX‐1 and COX‐2 inhibition. Recent studies on COX receptor expression in reptiles suggest that nonselective COX inhibitors may be more appropriate than more selective inhibitors in some reptiles, but few pharmacokinetic studies are available. The goal of this study was to determine single‐ and multidose (three consecutive days) pharmacokinetics of racemic ketoprofen administered intravenously and intramuscularly at 2 mg/kg in healthy juvenile loggerhead turtles (Caretta caretta). The S‐isomer is the predominant isomer in loggerhead sea turtles, similar to most mammals, despite administration of a 50:50 racemic mixture. Multidose ketoprofen administration demonstrated no bioaccumulation; therefore, once‐daily dosing will not require dose adjustment over time. S‐isomer pharmacokinetic parameters determined in this study were C_max of 10.1 μg/ml by IM injection, C₀ of 13.4 μg/ml by IV injection, AUC of 44.7 or 69.4 μg*hr/ml by IM or IV injection, respectively, and T½ of 2.8 or 3.6 hr by IM or IV injection, respectively. Total ketoprofen plasma concentrations were maintained for at least 12 hr above concentrations determined to be effective for rats and humans. A dose of 2 mg/kg either IM or IV every 24 hr is likely appropriate for loggerhead turtles.     

MIB‐1 immunoreactivity correlates with biologic behaviour in canine cutaneous melanoma

The growth fraction of 68 canine cutaneous melanomas was determined by immunostaining with MIB‐1, a monoclonal antibody to a Ki‐67 epitope that recognizes all proliferating cells. Fifty tumours were classified histologically as benign and 18 as malignant. The Ki‐67 proliferative index (percentage of positive cells over 500 neoplastic cells) was low (< 15%) in 55 cases and high ( 15%) in 13 cases. High Ki‐67 proliferative index and histological malignancy were both associated with significantly poorer 2‐year survival (P < 0.0001). However, the predictive value of the Ki‐67 proliferative index (97%) was higher than the predictive value of classical histology (91%). The evaluation of the growth fraction by the Ki‐67 proliferative index is highly predictive of the biological behaviour of canine cutaneous melanoma.     

Pilot study utilizing Fluorine‐18 fluorodeoxyglucose‐positron emission tomography/computed tomography for glycolytic phenotyping of canine mast cell tumors

The goal of this prospective pilot study was to use naturally occurring canine mast cell tumors of various grades and stages as a model for attempting to determine how glucose uptake and markers of biologic behavior are correlated. It was hypothesized that enhanced glucose uptake, as measured by 2‐[fluorine‐18]fluoro‐d ‐glucose‐positron emission tomography/computed tomography (F18 FDG PET‐CT), would correlate with histologic grade. Dogs were recruited for this study from a population referred for treatment of cytologically or histologically confirmed mast cell tumors. Patients were staged utilizing standard of care methods (abdominal ultrasound and three view thoracic radiographs), followed by a whole body F18 FDG PET‐CT. Results of the F18 FDG PET‐CT were analyzed for possible metastasis and standard uptake value maximum (SUV_max) of identified lesions. Incisional or excisional biopsies of the accessible mast cell tumors were obtained and histology performed. Results were then analyzed to look for a possible correlation between the grade of mast cell tumors and SUV_max. A total of nine animals were included in the sample. Findings indicated that there was a correlation between grade of mast cell tumors and SUV_max as determined by F18 FDG PET‐CT (p‐value = 0.073, significance ≤ 0.1). Based on the limited power of this study, it is felt that further research to examine the relationship between glucose utilization and biologic aggressiveness in canine mast cell tumors is warranted. This study was unable to show that F18 FDG PET‐CT was a better staging tool than standard of care methods.     

Overexpression of HER‐2 via immunohistochemistry in canine urinary bladder transitional cell carcinoma ‐ A marker of malignancy and possible therapeutic target

Transitional cell carcinoma (TCC) is the most commonly diagnosed neoplasm in the urinary bladder. Distant metastases to the regional lymph nodes, lungs, abdominal organs or bones are noted in up to 50% of dogs at time of death. Surgical excision is often not practical as TCC typically involve the trigone of the bladder and/or occurs multifocally throughout the bladder with field cancerization. Therapeutic approaches are very challenging and the requirement to evaluate alternative therapeutic protocols that may prolong survival times in dogs bearing these tumours is compelling. We assessed the immunohistochemical expression of HER‐2 in 23 cases of canine TCCs of the urinary bladder and compare it with non‐neoplastic urothelium in order to evaluate a rationale for targeted therapies and gene‐based vaccines. HER‐2 positivity was recorded in 13/23 (56%) neoplastic lesions. The receptor was significantly overexpressed in neoplastic than in non‐neoplastic samples (P = .015). According to our preliminary results, it would be of interest to further evaluate the role of HER‐2 in canine TCCs as a marker of malignancy and a therapeutic target for cancer vaccine and antibodies. Moreover, the significantly different overexpression of HER‐2 in TCCs than in non‐neoplastic urothelium further supports to investigate its role in the progression toward malignancy of non‐neoplastic lesions.     

Evaluation of the global DNA methylation in canine mast cell tumour samples by immunostaining of 5‐methyl cytosine

Cutaneous mast cell tumours (MCT) are the most common skin tumour in dogs, and to our knowledge, there are no previous studies regarding the global methylation of these tumours. DNA hypomethylation and hypermethylation have been described in several tumours and both mechanisms can lead to carcinogenesis. The purpose of this study was to evaluate the global DNA methylation in canine MCT. A total of 48 MCT samples were classified in grades 1, 2 and 3 or high‐grade or low‐grade. Monoclonal antibodies were used for the immunohistochemical detection of the 5‐methylcytosine. The immunostained nuclei were classified in strong, weak or negative pattern, and these were quantified in five distinct microscopic fields (40× objective) in each slide. The results showed that global DNA hypomethylation was predominant in grade 3, high‐grade, less differentiated MCT. These epigenetic changes in neoplastic mast cells warrant further detailed investigation aiming the establishment of tumour epigenetic therapies.     

Influence of E‐cadherin genetic variation in canine mammary tumour risk,clinicopathological features and prognosis

E‐cadherin is a cell adhesion molecule that participates in several cellular processes that guarantee the maintenance of structural and functional integrity of epithelial tissues. E‐cadherin plays an important role in mammary carcinogenesis, and various studies have demonstrated the effect of CDH1 genetic variation in risk, progression and biological behaviour of human breast cancer. Although there are some recognized genetic variations in canine CDH1 gene, their influence in canine mammary tumour development and progression has not been previously evaluated. In this study, we aim to assess the influence of CDH1 SNPs rs850805755, rs852280880 and rs852639930 in the risk, clinicopathological features and clinical outcome of canine mammary tumours. A case‐control study was conducted involving 206 bitches with mammary tumours and 161 bitches free of mammary neoplasia. CDH1 SNPs rs850805755 and rs852280880 were associated with a decreased risk and a later onset of mammary tumour development. Furthermore, these SNPs were related to the development of small size carcinomas, of low histological grade and low nuclear pleomorphism. SNP rs852639930 was associated with the development of small size tumours with a non‐infiltrative, non‐invasive growth pattern. Data from the present investigation demonstrate that these CDH1 genetic variants could have a protective role in canine mammary tumours, by being associated with low risk of tumour development, delayed onset of the disease and less aggressive clinicopathological features.