Novel genomic prognostic biomarkers for dogs with cancer.

BACKGROUND: Growing evidence from dogs and humans supports the abundance of mutation-based biomarkers in tumors of dogs. Increasing the use of clinical genomic diagnostic testing now provides another powerful data source for biomarker discovery. HYPOTHESIS: Analyzed clinical outcomes in dogs with cancer profiled using SearchLight DNA, a cancer gene panel for dogs, to identify mutations with prognostic value. ANIMALS: A total of 127 cases of cancer in dogs were analyzed using SearchLight DNA and for which clinical outcome information was available. METHODS: Clinical data points were collected by medical record review. Variables including mutated genes, mutations, signalment, and treatment were fitted using Cox proportional hazard models to identify factors associated with progression-free survival (PFS). The log-rank test was used to compare PFS between patients receiving and not receiving targeted treatment before first progression. RESULTS: Combined genomic and outcomes analysis identified 336 unique mutations in 89 genes across 26 cancer types. Mutations in 6 genes (CCND1, CCND3, SMARCB1, FANCG, CDKN2A/B, and MSH6) were significantly associated with shorter PFS. Dogs that received targeted treatment before first progression (n = 45) experienced significantly longer PFS compared with those that did not (n = 82, P = .01). This significance held true for 29 dogs that received genomically informed targeted treatment compared with those that did not (P = .05). CONCLUSION AND CLINICAL IMPORTANCE: We identified novel mutations with prognostic value and demonstrate the benefit of targeted treatment across multiple cancer types. These results provide clinical evidence of the potential for genomics and precision medicine in dogs with cancer.

[Questionnaire Survey on the Timing of Comprehensive Genomic Profiling for Advanced Recurrent Colorectal Cancer and Its Impact on Patients].

BACKGROUNDS: Comprehensive genomic profiling(CGP)has been covered by health insurance since June 2019. However, the clinical impact of CGP on patients with metastatic colorectal cancer(mCRC)remains unclear. To date, there are very limited reports regarding patient-oriented outcomes of CGP in mCRC. PATIENTS: A questionnaire was completed by patients with mCRC who had already received their CGP results after April 2021. Eight questions were posed, covering the degree of satisfaction and timing when CGP was conducted. RESULTS: Of the 51 patients with mCRC who had received their CGP test results by August 2021 in our department, 21 patients responded to our questionnaire. In total 66.7% patients with mCRC answered "(very)satisfied"with the CGP testing. 28.6% of the patients already knew about CGP testing before their local doctors informed them. Except for 3 patients who did not answer, 47.6% and 9.5% of patients with mCRC"agreed"and "moderately agreed"with the timing of the CGP test. CONCLUSION: Although most patients with mCRC failed to access promising new treatment via CGP, approximately half of the patients answered that they were satisfied with the CGP testing. Conversely, a few patients already knew about CGP testing before it was proposed by their doctors. Thus, the provision of information at an early stage is necessary.

[Educational Project of Self-Removal of the Intravenous Central Venous Access Port Guided by Patients with Advanced Colorectal Cancer Instructed by a Nurse in the University Hospital].

BACKGROUND: Fluorouracil infusion for 46±5h from the central venous(CV)port is required for mFOLFOX6, FOLFIRI, and FOLFOXIRI in patients with advanced colorectal cancer(CRC), followed by self-removal of the needle by patients. At our hospital, outpatients were instructed for self-removal of the needle, but the results were unsatisfactory. Therefore, instructions for self-removal of the needle from the CV port have been initiated at the patient ward since April 2019, making use of a hospital stay of 3 days. PATIENTS: We retrospectively enrolled patients with chemotherapy-introduced advanced CRC from the CV port who received instructions for self-removal of the needle in the outpatient department and ward between January 2018 and December 2021. RESULTS: Of all patients with advanced CRC, 21 received instructions at the outpatient department(OP)while 67 at patient ward(PW). Incidences of successful self-removal of the needle without the aid of others were similar: 47% in OP and 52% in PW(p=0.80). However, after several additional instructions involving their families, it was higher in PW than in OP(97.0 vs 76.1%, p=0.005). Incidences of successful self-removal of the needle without the aid of others in those aged≥75/<75, and≥65/<65 years were 0%/61.1%, and 35.4%/67.5%, respectively. OP was as a risk factor for failed self-removal of the needle in the logistic regression analysis(odds ratio: 11.19, 95%CI: 1.86- 67.30). CONCLUSION: Repeated instructions involving patients' families during the hospital stay improved the incidence of successful self-removal of the needle. Involvement of patients' families from the beginning may effectively improve self- removal of the needle, particularly in the elderly patients with advanced CRC.

Nosocomial outbreak of serious canine infectious tracheobronchitis (kennel cough) caused by canine herpesvirus infection.

Canine herpesvirus (CHV; Canid herpesvirus 1) is principally a perinatal pathogen of pregnant bitches and newborn pups and secondarily a respiratory tract pathogen of older pups and dogs. Infectious disease of the canine respiratory tract frequently occurs among dogs in groups, in which it is called " infectious tracheobronchitis" (ITB). Mortality from ITB is generally negligible, and the clinical importance of CHV as an ITB pathogen is considered to be low. The present report describes a novel ITB outbreak accompanied by death among aged dogs in an animal medical center. Most inpatient dogs had received medications that could induce immunosuppression. CHV was the only pathogen identified, and several CHV isolates were recovered in cell culture. No other viral pathogens or significant bacterial pathogens were found. Molecular and serological analyses revealed that the causative CHV isolates were from a single source but that none was a peculiar strain when the strains were compared with previous CHV strains. The virus had presumably spread among the dogs predisposed to infection in the center. The present results serve as a warning to canine clinics that, under the specific set of circumstances described, such serious CHV outbreaks may be expected wherever canine ITB occurs.

Activation of the PI3 kinase pathway by retinoic acid mediates sodium/iodide symporter induction and iodide transport in MCF-7 breast cancer cells.

Iodide uptake in the thyroid and breast is mediated by the sodium/iodide symporter (NIS). NIS activation is used for radioiodide imaging and therapeutic ablation of thyroid carcinoma. NIS is expressed in >70% of breast cancers but at a level insufficient for radioiodine treatment. All-trans retinoic acid (tRA) induces NIS gene expression and functional iodide uptake in human breast cancer cell lines and mouse breast cancer models. tRA usually regulates gene expression by direct interaction of RA receptor (RAR) with a target gene, but it can also act through nongenomic pathways. We report a direct influence of tRA treatment on the phosphoinositide 3-kinase (PI3K) signal transduction pathway that mediates tRA-induced NIS expression in MCF-7 breast cancer cells. MCF-7 cells express all three RAR isoforms, alpha, beta, and gamma, and RXRalpha. We previously identified RARbeta and RXRalpha as important for NIS induction by tRA. Treatment with LY294002, the PI3K inhibitor, or p85alpha knockdown with siRNA abolished tRA-induced NIS expression. Immunoprecipitation experiments and glutathione S-transferase pull-down assay showed a direct interaction between RARbeta2, RXRalpha, and p85alpha. RA also induced rapid activation of Akt in MCF-7 cells. Treatment with an Akt inhibitor or Akt knockdown with siRNA reduced NIS expression. These findings indicate that RA induction of NIS in MCF-7 cells is mediated by rapid activation of the PI3K pathway and involves direct interaction with RAR and retinoid X receptor. Defining these mechanisms should lead to methods to further enhance NIS expression, as well as retinoid targets that influence growth and differentiation of breast cancer.

Retinoic acid stimulation of the sodium/iodide symporter in MCF-7 breast cancer cells is mediated by the insulin growth factor-I/phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase signaling pathways.

CONTEXT: All-trans retinoic acid (tRA) induces differentiation in MCF-7 breast cancer cells, stimulates sodium/iodide symporter (NIS) gene expression, and inhibits cell proliferation. Radioiodine administration after systemic tRA treatment has been proposed as an approach to image and treat some differentiated breast cancer. OBJECTIVE: The objective of this work was to study the relative role of genomic and nongenomic pathways in tRA stimulation of NIS expression in MCF-7 cells. DESIGN: We inspected the human NIS gene locus for retinoic acid-responsive elements and tested them for function. The effects of signal transduction pathway inhibitors were also tested in tRA-treated MCF-7 cells and TSH-stimulated FRTL-5 rat thyroid cells, followed by iodide uptake assay, quantitative RT-PCR of NIS, and cell cycle phase analysis. RESULTS: Multiple retinoic acid response elements around the NIS locus were identified by sequence inspection, but none of them was a functional tRA-induced element in MCF-7 cells. Inhibitors of the IGF-I receptor, Janus kinase, and phosphatidylinositol 3-kinase (PI3K), significantly reduced NIS mRNA expression and iodide uptake in tRA-stimulated MCF-7 cells but not FRTL-5 cells. An inhibitor of p38 MAPK significantly reduced iodide uptake in both tRA-stimulated MCF-7 cells and TSH-stimulated FRTL-5 cells. IGF-I and PI3K inhibitors did not significantly reduce the basal NIS mRNA expression in MCF-7 cells. Despite the chronic inhibitory effects on cell proliferation, tRA did not reduce the S-phase distribution of MCF-7 cells during the period of NIS induction. CONCLUSION: The IGF-I receptor/PI3K pathway mediates tRA-stimulated NIS expression in MCF-7 but not FRTL-5 thyroid cells.

Role of beta1 integrins in adhesion of canine mastocytoma cells to extracellular matrix proteins.

The interactions of tumor cells with the extracellular matrix (ECM) are a crucial step in invasion and metastasis. Integrins are adhesive molecules forming heterodimers with alpha and beta subunits that play a definitive role in these interactions. In this study, mastocytoma (mast cell tumor: MCT) cell-ECM interaction was investigated using 3 canine MCT cell lines: CM-MC (originating from cutaneous MCT), VI-MC (originating from intestinal MCT), and CoMS (originating from oral MCT). Flow cytometric analysis showed that all cells highly expressed the integrin beta1 and alpha1 through alpha5 subunits and that they moderately expressed the alpha6 subunit. In adhesion studies, CoMS weakly but spontaneously adhered to fibronectin (FN), which was enhanced by phorbol ester (TPA), while CM-MC and VI-MC required cell activation by TPA to adhere to FN. Anti-beta1 and alpha5 integrin antibodies strongly inhibited cell adhesion to FN in CM-MC and CoMS and moderately inhibited cell adhesion in VI-MC. Only VI-MC adhered to laminin (LN) under activation by TPA. Anti-beta1 integrin antibodies strongly inhibited cell adhesion to LN, but all anti-alpha integrin antibodies failed to inhibit cell adhesion to LN. No cells adhered to collagen types I and IV. Canine MCT cells from different origins expressed similar integrin patterns; however, there were some differences in adhesive behavior in response to various ECM proteins and activating stimuli.

Retinoids induce growth inhibition and apoptosis in mast cell tumor cell lines.

Retinoids are well recognized as promising antitumor agents in humans. However, there have only been a few reports about the effect of retinoids in canine cancers. To investigate the antitumor effect of retinoids on mast cell tumors (MCT), inhibitory effect on cell growth and induction of apoptosis were examined in vitro. Although sensitivity of these cells differed among the cells, the growth of three MCT cell lines (CoMS, CM-MC and VI-MC) were inhibited dose dependently when they were treated with retinoids. FACS analysis of PI-stained nuclei revealed an apoptotic fraction in CM-MC cells about 30% when treated with retinoids, while those of control cells were less than 5%. Caspase-3 activation was observed after retinoid treatment in CM-MC cells. This was confirmed by inhibiting the retinoid-induced apoptosis using the pan-caspase inhibitor, ZVAD-FMK. Both retinoid receptors, RARs and RXRs, were detected by immunoprecipitation followed by western blot analysis in all the three MCT cells. These data suggests that retinoids inhibit the growth of MCTs partly through apoptosis, and this growth inhibition by retinoids may be mediated by RARs and RXRs. We conclude that retinoid may be a potential adjunctive chemotherapeutic agent for the treatment of canine MCT.

Relationship between retinoic acid receptor alpha gene expression and growth-inhibitory effect of all-trans retinoic acid on canine tumor cells.

Retinoids show antitumor effects on human acute promyelocytic leukemia and other tumors via retinoid receptors. In dogs, the role of retinoid receptors in inhibiting tumor development remains unclear. To evaluate the correlation between the degree of expression of retinoic acid receptor alpha (RARalpha) mRNA and the antiproliferative effects of all-trans retinoic acid (ATRA) treatments, expression analysis of RARalpha mRNA and cell growth inhibition assay were performed on 17 established canine tumor cell lines, including 6 mammary gland tumor (MGT) cell lines, 3 osteosarcoma cell lines, 5 melanoma cell lines, and 3 mast cell tumor (MCT) cell lines. Among the cell lines investigated, all 3 MCT cell lines showed high expression of RARalpha, and the most effective cell growth inhibition was observed in ATRA-treated MCT cell lines. However, remarkable antiproliferative effects of ATRA treatments were not observed on other tumor cell lines with moderate or low RARalpha mRNA expression. As a result of the relationship between RARalpha mRNA expression and ATRA treatment with regression analysis, statistically significant correlation was suggested. Furthermore, real-time quantitative polymerase chain reaction analysis of RARalpha was performed on MCT tissue samples of dogs with spontaneous disease, and 5 of 9 tissues showed high expression. These results suggest that ATRA may be an effective antitumor agent for MCT in dogs, and that prior measurement of expression of RARalpha mRNA may be a good indicator of the effectiveness of ATRA treatment.

Identification and characterization of a canine highly similar to retinoic acid receptor alpha.

A canine highly similar to retinoic acid receptor alpha (canine HS-RARa) cDNA was isolated from the spleen tissue. A database search and the alignment revealed that the canine cDNA was most similar to highly similar type of human RARa and was named canine HS-RARa. The expression of the genes encoding RARa in the dog was the highest in the testis and moderate in the blood, lymph node, mammary gland, pancreas, salivary gland, spleen, thyroid gland, tonsil and uterus. The nucleotide sequence encoded the 462-amino acid containing the conserved sequence motif of RARa. Though the amino acid sequences were well-conserved among species, some unique arrangements were observed within each class. In the phylogenetic analysis, each species separated according to their class. In the branch of mammals, the dog is in the cluster of humans, mice and western wild mice. However, hamsters and rats formed another branch.

Differential regulation of sodium/iodide symporter gene expression by nuclear receptor ligands in MCF-7 breast cancer cells.

The sodium/iodide symporter (NIS) mediates iodide uptake in lactating breast tissue and is expressed in some breast cancers. We have previously demonstrated that all-trans retinoic acid (tRA) stimulates NIS gene expression and the selective cytotoxic effect of beta-emitting radioiodide-131 ((131)I) in both in vitro and in vivo MCF-7 breast cancer cell systems. We studied the ability of natural and synthetic retinoids, in combination with other nuclear receptor ligands, to achieve greater and more sustained induction of NIS in MCF-7 cells and enhance (131)I-mediated cytotoxicity. Selective stimulation of retinoic acid receptor (RAR) beta/gamma produced marked NIS induction; and selective stimulation of RARalpha, RARgamma, or retinoid X receptor produced more modest induction. Maximal NIS induction was seen with 9-cis retinoic acid and AGN190168, a RAR beta/gamma-agonist. Dexamethasone (Dex), but not the other nuclear receptor ligands, in combination with tRA synergistically induced iodide uptake and NIS mRNA expression, predominantly by prolonging NIS mRNA half-life. The addition of Dex reduced the EC(50) of tRA for NIS stimulation to approximately 7%, such that 10(-7) m tRA with addition of Dex enhanced iodide uptake and selective cytotoxicity of (131)I greater than 10(-6) m tRA alone. AGN190168 combined with Dex synergistically increased iodide uptake and significantly prolonged induction (5 d) of iodide uptake compared with that induced by the combination of tRA/Dex or 9-cis retinoic acid/Dex. The addition of Dex reduced the effective dose of retinoid and prolonged the induction of NIS, especially with AGN190168, suggesting higher efficacy of (131)I after combination treatment.

Establishment and characterization of four canine melanoma cell lines.

Four canine melanoma cell lines were established from the subcutaneous, oral gingival and mucosal melanoma tissues at the primary and metastatic sites. These cell lines were designated as CMeC-1, CMeC-2, KMeC and LMeC. The cells were spindles in shape, similar to that of primary tumor cells. The doubling times of these cells ranged from 34.1 +/- 5.61 to 57.9 +/- 3.28 hr and their chromosome number ranged from 46 to 80. When transplanted into nude mice, CMeC-1 and LMeC produced tumors, whereas CMeC-2 and KMeC did not. The morphology of the tissue formed by xenotransplantation of these cells was similar to their primary tumors.

Molecular cloning of the canine telomerase reverse transcriptase gene and its expression in neoplastic and non-neoplastic cells.

OBJECTIVE: To perform molecular cloning of the canine telomerase reverse transcriptase (TERT) gene and determine its expression in neoplastic and non-neoplastic cells. SAMPLE POPULATION: 9 canine tumor cell lines derived from various neoplasms, 16 primary canine tumors, and tissues from 15 normal canine organs. PROCEDURE: Tumor cell lines were derived from canine tumors that included osteosarcoma, mammary gland adenocarcinoma, melanoma, acute lymphoblastic leukemia, lymphoma, and mastocytoma and a canine primary fibroblast culture. Canine TERT complementary DNA (cDNA) was amplified by use of polymerase chain reaction (PCR) and sequenced. Expression of TERT mRNA was examined by reverse transcription (RT)-PCR assay. Telomerase activity was measured by use of the telomeric repeat amplification protocol assay. RESULTS: The canine TERT cDNA clone was 237 base pairs in length and contained a central region encoding the reverse transcriptase motif 2. Expression of TERT mRNA was detected in canine tumor cell lines that had telomerase activity but not in telomerase-negative canine primary fibroblasts. The TERT mRNA was detected in 13 of 16 canine tumor tissues and several normal tissues such as liver, ovary, lymph node, and thymus. A significant correlation between TERT expression level and telomerase activity was noted. CONCLUSIONS AND CLINICAL RELEVANCE: Expression of TERT mRNA was closely associated with telomerase activity in neoplastic cells as well as some non-neoplastic cells from dogs. In addition to telomerase activity, expression of TERT mRNA can be used as a marker of tumor cells.

The expression of sialyl Lewis X in canine and feline mammary gland tumors.

The expression of sialyl Lewis X (sLe(x)) in 93 canine and 15 feline mammary gland tumors (MGT) obtained by surgical resection at Veterinary Medical Center, the University of Tokyo was examined by immunohistochemistry. Their clinicopathological features and prognosis were also reviewed. Approximately 60% of MGT tissues showed sLe(x) positive expressions, while all normal mammary gland tissues were negative. However, its expression was not correlated with clinicopathological features and prognosis significantly. This study suggests that sLe(x) may be a tumor-associated antigen in canine and feline MGTs.

Effect of natural and synthetic retinoids on the proliferation and differentiation of three canine melanoma cell lines.

The effect of two natural retinoids and synthetic retinoids with or without retinoid synergists on the proliferation and differentiation of 3 melanoma cell lines were investigated in vitro. No retinoids showed significant growth inhibitory effect on these cell lines when used alone, however, cell differentiation and significant growth inhibition were observed when treated with a combination of retinoids and a retinoid synergist. This study may suggest that, though the cells showed low susceptibilities when retinoids were treated alone, the combination of retinoids and a retinoid synergist may be effective to control the growth of canine melanoma cell lines.