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Nodal metastasis is a negative prognostic factor in dogs with mast cell tumours (MCTs), thus early detection enables more informed decision-making and provides valuable prognostic information. The aim of this study is to assess the concordance between histopathologic findings of LNs and cytology and flow cytometry (FC), respectively, and to evaluate the ability of FC to differentiate between metastatic (HN2-HN3) and non-metastatic (HN0-HN1) LNs. Overall, 117 LNs from 64 dogs with first occurring MCTs were submitted for cytology, histology and FC. LNs were cytologically and histologically classified according to Krick and Weishaar systems, respectively. Using FC, mast cells (MCs) were identified as IgE+ CD117+ CD5- CD21- cells and quantified as a percentage. When compared with histologic classification, cytology showed an accuracy of 88.2% in distinguishing between metastatic and non-metastatic LNs but did not detect 25.3% of metastatic cases. FC revealed an increase in the median percentages of MCs across histologic classes, progressing from HN0 to HN3. ROC curves pinpointed 0.3% as the optimal cut-off for distinguishing between metastatic and non-metastatic LNs, with an accuracy of 84.3%. A 1.1% cut-off proved valuable in identifying HN3 LNs. The combined interpretation of cytology and FC increased accuracy to 92.2%. An algorithm for guiding the combined interpretation of cytology and FC is suggested based on these findings. In conclusion, FC proves beneficial in enhancing the early detection of metastatic LNs, particularly when utilised alongside cytology. Histopathology remains essential for confirmation, enabling the discrimination of HN classes or, in doubtful cases, for the detection or exclusion of nodal metastases., (© 2024 John Wiley & Sons Ltd.)

Canine oral malignant melanoma (COMM) is the most common neoplasm in the oral cavity characterized by local invasiveness and high metastatic potential. Hypoxia represents a crucial feature of the solid tumor microenvironment promoting cancer progression and drug resistance. Hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, vascular endothelial growth factor A (VEGF-A), glucose transporter isoform 1 (GLUT1), C-X-C chemokine receptor type 4 (CXCR4), and carbonic anhydrase IX (CAIX), are the main regulators of the adaptive response to low oxygen availability. The prognostic value of these markers was evaluated in 36 COMMs using immunohistochemistry. In addition, the effects of cobalt chloride-mediated hypoxia were evaluated in 1 primary COMM cell line. HIF-1α expression was observed in the nucleus, and this localization correlated with the presence or enhanced expression of HIF-1α-regulated genes at the protein level. Multivariate analysis revealed that in dogs given chondroitin sulfate proteoglycan-4 ( CSPG4 ) DNA vaccine, COMMs expressing HIF-1α, VEGF-A, and CXCR4 were associated with shorter disease-free intervals (DFI) compared with tumors that were negative for these markers ( P = .03), suggesting hypoxia can influence immunotherapy response. Western blotting showed that, under chemically induced hypoxia, COMM cells accumulate HIF-1α and smaller amounts of CAIX. HIF-1α induction and stabilization triggered by hypoxia was corroborated by immunofluorescence, showing its nuclear translocation. These findings reinforce the role of an hypoxic microenvironment in tumor progression and patient outcome in COMM, as previously established in several human and canine cancers. In addition, hypoxic markers may represent promising prognostic markers, highlighting opportunities for their use in therapeutic strategies for COMMs., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Myeloid sarcoma (MS) is a solid tumor of granulocytic origin with extramedullary localization. This tumor is rare in humans and animals. The diagnostic approach is heterogeneous, and the definitive diagnosis may be difficult to achieve. Primary MS has never been described as a spontaneous neoplasm in companion dogs. Two purebred and 1 mixed-breed dogs, 6- to 11-year-old, developed round cell tumors in the mediastinum, lymph nodes (LNs) and tonsils, and LNs, respectively. Granulocytic origin and exclusion of lymphoid lineage were confirmed by flow cytometry, supported by immunohistochemistry or immunocytochemistry. Pivotal to the diagnosis were positive labeling for myeloid (CD11b, CD14) and hematopoietic precursors (CD34) markers, along with negative labeling for lymphoid markers. Blood and bone marrow infiltration were not detected at initial diagnosis, excluding acute myeloid leukemia. The behavior of these tumors was aggressive, resulting in poor clinical outcomes, even when chemotherapy was attempted., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), is a rare but potentially fatal complication following allogenic hematopoietic cell transplantation (allo-HCT). Timely identification of SOS/VOD to allow for prompt treatment is critical, but identifying a VOD-predictive biomarker remains challenging. Given the pivotal role of endothelial dysfunction in SOS/VOD pathophysiology, the CECinVOD study prospectively evaluated levels of circulating endothelial cells (CECs) in patients undergoing allo-HCT with a myeloablative conditioning (MAC) regimen to investigate the potential of CEC level in predicting and diagnosing SOS/VOD. A total of 150 patients from 11 Italian bone marrow transplantation units were enrolled. All participants were age >18 years and received a MAC regimen, putting them at elevated risk of developing SOS/VOD. Overall, 6 cases of SOS/VOD (4%) were recorded. CECs were detected using the Food and Drug Administration-approved CellSearch system, an immunomagnetic selection-based platform incorporating ferrofluid nanoparticles and fluorescent-labeled antibodies, and were defined as CD146+, CD105+, DAPI+, or CD45-. Blood samples were collected at the following time points: before (T0) and at the end of conditioning treatment (T1), at neutrophil engraftment (T2), and at 7 to 10 days postengraftment (T3). For patients who developed VOD, additional samples were collected at any suspected or proven VOD onset (T4) and weekly during defibrotide treatment (T5 to T8). A baseline CEC count >17/mL was associated with an elevated risk of SOS/VOD (P = .04), along with bilirubin level >1.5 mg/mL and a haploidentical donor hematopoietic stem cell source. Postconditioning regimen (T1) CEC levels were elevated (P = .02), and levels were further increased at engraftment (P < .0001). Additionally, patients developing SOS/VOD after engraftment, especially those with late-onset SOS/VOD, showed a markedly higher relative increase (>150%) in CEC count. Multivariate analysis supported these findings, along with a high Endothelial Activation and Stress Index (EASIX) score at engraftment (T2). Finally, CEC kinetics corresponded with defibrotide treatment. After the start of therapy (T4), CEC levels showed an initial increase in the first week (T5), followed by a progressive decrease during VOD treatment (T6 and T7) and a return to pre-SOS/VOD onset levels at resolution of the complication. This prospective multicenter study reveals a low incidence of SOS/VOD in high-risk patients compared to historical data, in line with recent reports. The results from the CECinVOD study collectively confirm the endothelial injury in allo-HCT and its role in in the development of SOS/VOD, suggesting that CEC level can be a valuable biomarker for diagnosing SOS/VOD and identifying patients at greater risk of this complication, especially late-onset SOS/VOD. Furthermore, CEC kinetics may support treatment strategies by providing insight into the optimal timing for discontinuing defibrotide treatment., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Osteosarcoma is the most common malignant primary bone cancer, but it is infrequently reported in cats. Feline appendicular osteosarcoma typically exhibits good prognosis when treated with surgery alone. A retrospective multi-institutional study was conducted to identify possible prognostic factors. Cats diagnosed with appendicular osteosarcoma were included if initial staging and follow-up information were available. Data including signalment, tumour characteristics, treatment modalities, and survival outcomes were collected and analysed. Fifty-six cats were included; the femur was the most frequently affected bone. Eight cats had distant metastasis at admission and an additional 9 developed metastatic disease during follow-up, resulting in an overall metastatic rate of 30%. Forty-nine (87.5%) cats underwent surgery, and 4 also received adjuvant chemotherapy. Among operated cats, median time to local progression (TTLP), time to distant progression and tumour-specific survival (TSS) were not reached. One- and 2-year survival rates were 66% and 55%, respectively. Seven (12.5%) cats received no treatment; 1- and 2-year survival rates were 25% and 0%, respectively. Operated cats had significantly longer TTLP (P < .001) and TSS (P = .001) compared with non-operated cats. Among operated cats, young age negatively impacted local tumour progression, while the presence of distant metastasis at diagnosis was associated with a higher risk of tumour-related death. This study reaffirms the good prognosis for cats with appendicular osteosarcoma undergoing surgery, but sheds light on some additional factors to consider. Accurate initial staging is recommended, as the metastatic rate may exceed many previous estimations. Surgery substantially extends survival time, whereas the role of chemotherapy remains uncertain., (© 2024 John Wiley & Sons Ltd.)

Autoimmune diseases (ADs) represent a heterogeneous group of conditions affecting 5-10% of the global population. In recent decades, hematopoietic stem cell transplant (HSCT), mainly autologous, has been successfully adopted to treat patients affected by severe/refractory ADs. In this context malnutrition has a detrimental impact on relapse, mortality, infection rate, engraftment, long-term survival, and prolongation of hospitalization. However, in this population, the management of nutrition should be improved since nutritional assessment is partially performed in routine clinical practice. A panel of nurses and physicians from the European Society for Blood and Marrow Transplantation (EBMT) reviewed all available evidence based on current literature and expert practices from centers with extensive experience in HSCT for ADs, on the nutritional management of ADs patients during HSCT procedure. In this context, adequate nutritional status predicts a better response to treatment and improves quality of life. Herein, a systematic and comprehensive monitoring of nutritional status before, during and after HSCT, with adequate nutritional support in the case of ADs patients, in addition to assessing the dietary requirements associated with HSCT has been covered. Moreover, given the singularity of each AD, the underlying disease should be considered for an appropriate approach. The management and evaluation of nutritional status must be carried out by a multidisciplinary team to assess the needs, monitor the effectiveness of each intervention, and prevent complications, especially in complex situations as patients affected by ADs., Competing Interests: RG discloses speaking honoraria from Biotest, Pfizer, Medac, Neovii and Magenta. TA received study support from Amgen, Janssen and honoraria from Neovii, GSK, Astra-Zeneca, Abbvie. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Gandossi, Jessop, Hahn, Heininger, Henes, Radaelli, Carmagnola, Morello, Renica, Bertulli, Lazzari, Kenyon, Alexander, Domenech and Greco.)

The rise of Vancomycin-resistant enterococci (VRE) strains among cellular therapy recipients raises concerns due to increased morbidity, mortality, and hospitalization costs, particularly impacting transplanted patients with diminished survival expectations. Recent research linking lactose to Enterococcus growth and graft-versus-host disease (GVHD) emphasizes the need for data on reducing lactose in the diets of VRE-carrying patients, especially in cellular therapy contexts like CAR-T or allogeneic hematopoietic stem cell transplantation. Responding to elevated VRE positivity rates in rectal swabs among patients in our BMT Unit, a unique nutritional strategy was implemented, introducing lactose-free milk and strictly enforcing lactose-free diets. This approach resulted in a significant reduction in VRE carriers, with a 16% positivity rate in the Lactose Group versus 3.6% in the Lactose-Free Group, as of June 2023. These results indicate the potential efficacy of this innovative nutritional strategy in high-risk departments, such as BMT Units and Intensive Care Units, with implications for reducing isolation strategies and inappropriate antibiotic use in cases of VRE colonization., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Malnutrition in allogeneic stem cell transplant (allo-SCT) is associated with poor outcomes. Supplementation with Foods for Special Medical Purposes may be a valid alternative to enteral nutrition or total parental nutrition to reduce malnutrition in allo-SCT. In this study, 133 patients consecutively allo-transplanted were assessed for nutritional status by Patient- Generated Subjective Global Assessment (PG-SGA) and supplemented with TGF-beta enriched Food for Special Medical Purposes (TGF-FSMP). PG-SGA, gold standard for nutritional assessment in oncologic patients, was assessed at admission and on day 0, +7, +14, +21, and + 28 from transplant and categorized as follows: A = good nutritional status; B = moderate malnutrition; C = severe malnutrition. TGF-FSMP (Modulen-IBD) is currently used in Inflammatory Bowel Diseases (IBD) as primary nutritional support and in this study the dose was calculated according to BMI and total daily energy expenditure (TDEE). The patients assuming ≥50% of the prescribed TGF-FSMP dose were classified in Group A; the patients who received < 50% were included in Group B per protocol. The primary endpoint of the study was the assessment of the malnourished patients in Group A and B at day+28 after transplantation, according to the criteria of PG-SGA C categorization. At day +28 after transplant: i) patients in Group A were significantly less severely malnourished than patients in the Group B (21/76,28% vs 42/53, 79% respectively, OR 2.86 - CI 1.94-4.23 -, p = 0.000); ii) the incidence of severe (MAGIC II-IV) aGVHD and of any grade gastrointestinal (GI) aGVHD was higher in Group B than in Group A, (43% vs 21% p = 0.003) and (34.5% vs 9.2% p = 0.001); iii) Pneumonia was more frequent in the malnourished patients of Group B than in well/moderate nourished patients of Group A (52.7% vs 27.6% p = 0.002). In group A parenteral nutrition was avoided more frequently than in group B (67.5% vs 33.3% p = 0.000) and a median hospital stay of 27 days in comparison to 32 was reported (p = 0.006). The estimated median overall survival (OS) of the population was 33 months in Group A and 25.1 months in group B (p = 0.03). By multivariate and ANN analysis, TGF-FSMP TR < 50% assumption was significantly correlated with malnutrition, severe and GI aGVHD, pneumonia and reduced OS., Competing Interests: Declaration of Competing Interest The authors declare that there are not conflicting interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The high mortality rate of osteosarcoma (OSA) patients highlights the requirement of alternative strategies. The young age of patients, as well as the rarity and aggressiveness of the disease, limits opportunities for the robust testing of novel therapies, suggesting the need for valuable preclinical systems. Having previously shown the overexpression of the chondroitin sulfate proteoglycan (CSPG)4 in OSA, herein the functional consequences of its downmodulation in human OSA cells were evaluated in vitro, with a significant impairment of cell proliferation, migration, and osteosphere generation. The potential of a chimeric human/dog (HuDo)-CSPG4 DNA vaccine was explored in translational comparative OSA models, including human xenograft mouse models and canine patients affected by spontaneous OSA. The adoptive transfer of HuDo-CSPG4 vaccine-induced CD8 + T cells and sera in immunodeficient human OSA-bearing mice delayed tumor growth and metastasis development. HuDo-CSPG4 vaccination resulted safe and effective in inducing anti-CSPG4 immunity in OSA-affected dogs, which displayed prolonged survival as compared to controls. Finally, HuDo-CSPG4 was also able to induce a cytotoxic response in a human surrogate setting in vitro. On the basis of these results and the high predictive value of spontaneous OSA in dogs, this study paves the way for a possible translation of this approach to humans., Competing Interests: Declaration of interests None of the authors have a conflict of interest to declare., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Hematological indices play a prognostic role in human osteosarcoma (OSA), but data are limited in dogs. The aim of this retrospective multicentric cohort study was to investigate the prognostic significance of pre-operative hematological/inflammatory indices in a cohort of client-owned dogs with appendicular OSA receiving standardized treatment. Cut-offs associated with progression-free survival (PFS) for pre-operative hematological values/ratios were established using the minimal p -value approach. Historical prognostic factors were also assessed. Statistical analyses were performed for the whole population and after the exclusion of sighthounds. Fifty-nine dogs were included (13 were sighthounds). Multivariable analysis revealed that a low neutrophil count (<4.37 × 10 9 /L, HR0.28, CI 95% 0.13-0.61, p = 0.001), a high red blood cell count (≥7.91, HR3.5, CI 95% 1.56-7.9, p = 0.002), and a proximal humerus location (HR3.0, CI 95% 1.48-6.1, p = 0.002) were associated with shorter PFS. In the sighthound-only population, only OSA location was significantly associated with PFS in univariable analysis. When sighthounds were excluded, a low neutrophil count, a low monocyte count, and a proximal humerus location were associated with shorter PFS, in multivariable analysis. Neutrophil count and possibly monocyte and red blood cell counts can be useful prognostic markers in canine OSA treated with amputation and adjuvant carboplatin. However, not all indices are appropriate in sighthounds.

Saliva is an irritant of the subcutaneous tissue, thus causing the development of a non-epithelial reactive pseudocapsule. Metaplastic ossification of the pseudocapsule is a condition rarely described in the veterinary literature. The main causes of calcification are trauma, tumours, various chronic inflammatory conditions and fibrodysplasia ossificans progressiva . The aim of the present case series was to describe three dogs affected by a calcified salivary mucocele. The medical records of dogs affected by a cervical sialocele were retrospectively evaluated, and three cases met the inclusion criteria. All the dogs in this study were referred to the Veterinary Teaching Hospital (VTH) of the Department of Veterinary Sciences of the University of Turin (Turin, Italy) for a large solid mass in the intermandibular region. The diagnosis of a mucocele was confirmed clinically by centesis and by radiography or CT. Complete excision of both the pseudocyst and the ipsilateral mandibular/monostomatic sublingual salivary gland was performed in all cases. The histological report showed large areas of bone metaplasia within the pseudocapsule and chronic sialadenitis. Based on this limited case series, complete excision of the pseudocyst and a concurrent sialoadenectomy provided an effective treatment for this rare salivary mucocele disorder.

Background: Minimal residual disease (MRD) monitoring is an important tool to optimally address post-transplant management of acute myeloid leukemia (AML) patients., Methods: We retrospectively analyzed the impact of bone marrow CD34+ molecular chimerism and WT1 on the outcome of a consecutive series of 168 AML patients submitted to allogeneic stem cell transplantation., Results: The cumulative incidence of relapse (CIR) was significantly lower in patients with donor chimerism on CD34+ cells ≥ 97.5% and WT1 < 213 copies/ABL x 10^4 both at 1 st month (p=0.008 and p<0.001) and at 3 rd month (p<0.001 for both). By combining chimerism and WT1 at 3 rd month, 13 patients with chimerism < 97.5% or WT1 > 213 showed intermediate prognosis. 12 of these patients fell in this category because of molecular chimerism < 97.5% at a time-point in which WT1 was < 213., Conclusions: Our results confirm that lineage-specific molecular chimerism and WT1 after allo-SCT (1 st and 3 rd month) are useful MRD markers. When considered together at 3 rd month, CD34+ molecular chimerism could represent an earlier predictor of relapse compared to WT1 . Further studies are necessary to confirm this preliminary observation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Malagola, Polverelli, Beghin, Bolda, Comini, Farina, Morello, Radici, Accorsi Buttini, Bernardi, Re, Leoni, Bonometti, Brugnoni, Lanfranchi and Russo.)

Background and Aims: Telemonitoring is increasingly used in the management of IBD patients. We investigated the agreement between patients and physicians on scores of disease activity and burden., Methods: Consecutive outpatients at one IBD clinic were recruited between February and December 2021. Enrolled patients completed a questionnaire for disease activity (Harvey-Bradshaw Index [HBI] for Crohn's disease or Simple Clinical Colitis Activity Index [SCCAI] for ulcerative colitis) and a test of disease burden (Pictorial Representation of Illness and Self Measure [PRISM]). They did the tests within 5 days of an outpatient visit, working independently on IBD Tool, a new web-based telemonitoring application. Concomitantly, the senior and junior physicians who examined them completed the same tests. The agreement was tested for every pair of scores., Results: Five hundred and sixty patients (289 Crohn's disease; 271 ulcerative colitis) completed disease questionnaires on IBD Tool (in total, 742 times). By Spearman's correlation, the agreement was substantial both for HBI (rho 0.685-0.837) and SCCAI (rho 0.694-0.888) for comparisons between patients, junior and senior physicians. The agreement was moderate-to-substantial for PRISM (rho 0.406-0.725) for the same comparisons. The correlation between disease activity (HBI/SCCAI) and PRISM scores was substantial for senior (rho 0.757-0.788) or junior (rho 0.746-0.753) physicians and moderate for patients (rho 0.458-0.486). The median PRISM score difference was 2.3-1.6 points lower between patients and senior-junior physicians., Conclusion: Agreement between IBD patients and physicians was substantial for disease activity and moderate for disease impact. The inclusion of disease burden scoring in telemonitoring platforms provides important information for the management of IBD patients.Study highlightsWhat IS known•Continuous response to treatments and patient-reported outcomes became an essential goal for IBD patient management.•The use of tele-monitoring and eHealth technologies allows for regular disease assessments and for managing more efficiently IBD patients; disease questionnaires and tests are key to support eHealth tools.What is new here•Agreement between IBD patients and physicians was substantial for disease activity and moderate for disease burden, while agreement among junior and senior physicians was substantial for both.•PRISM performs as well for ulcerative colitis as for Crohn's patients.•The inclusion of disease burden tests might add to eHealth platforms valuable information, complemental to disease activity questionnaires.

Objectives: To evaluate the feasibility and the complications following single or double random mucosal rotating (transposition or interpolation) flaps for the closure of rostral to mid maxillary defects in dogs., Materials and Methods: Medical records of dogs treated with single or double random mucosal rotating flaps after maxillectomy for oral lesions or traumatic loss of tissue, were evaluated. Clinical findings, surgery performed, outcome and postoperative complications (major and minor) were extracted., Results: Twenty-six client-owned dogs were retrospectively included. Dogs underwent maxillectomy for canine acanthomatous ameloblastomas (9), oral squamous cell carcinomas (4), peripheral odontogenic fibromas (4), oral melanomas (3), oral fibrosarcomas (2), dentigerous cysts (2) and oral osteosarcoma (1) and trauma resulting in an oronasal fistula (1). Twenty-three dogs underwent a single transposition or interpolation flap and three dogs were treated with a double transposition flap. Postoperative complications, including dehiscence or flap necrosis, occurred in six dogs., Clinical Significance: Random mucosal rotating (transposition or interpolation) flaps are versatile when used to close rostral maxillary defects in dogs. Postoperative complications appear to be more likely when these flaps are used to close mid maxillary defects., (© 2022 The Authors. Journal of Small Animal Practice published by John Wiley & Sons Ltd on behalf of British Small Animal Veterinary Association.)

Bacteriophages have been identified as a potential treatment option to treat lung infection in the context of antibiotic resistance. We performed a preclinical study to predict the efficacy of delivery of bacteriophages against Pseudomonas aeruginosa (PA) when administered via nebulization during mechanical ventilation (MV). We selected a mix of four anti-PA phages containing two Podoviridae and two Myoviridae, with a coverage of 87.8% (36/41) on an international PA reference panel. When administered via nebulization, a loss of 0.30-0.65 log of infective phage titers was measured. No difference between jet, ultrasonic and mesh nebulizers was observed in terms of loss of phage viability, but a higher output was measured with the mesh nebulizer. Interestingly, Myoviridae are significantly more sensitive to nebulization than Podoviridae since their long tail is much more prone to damage. Phage nebulization has been measured as compatible with humidified ventilation. Based on in vitro measurement, the lung deposition prediction of viable phage particles ranges from 6% to 26% of the phages loaded in the nebulizer. Further, 8% to 15% of lung deposition was measured by scintigraphy in three macaques. A phage dose of 1 × 10 9 PFU/mL nebulized by the mesh nebulizer during MV predicts an efficient dose in the lung against PA, comparable with the dose chosen to define the susceptibility of the strain.

Compartmental excision consists of the complete resection of an anatomic district in which specific structures act as a barrier to local tumour invasion. It is a well-established procedure in human medicine, while only a few reports are available in veterinary medicine. The aim of this study was to describe complete muscle resection in 3 dogs affected by different intramuscular sarcomas. The clinical outcome was also reported. Medical records were searched, including preoperative diagnostic findings, compartmental excision, histologic diagnosis, and outcome. Three dogs fit the inclusion criteria, which had a sarcoma confined to a single muscular belly (semitendinosus, biceps, and splenius capitis muscles). Complete excision of the affected muscle was performed in all cases. One dog showed moderate lameness in the immediate postoperative period, resulting from the dorsal lifting of the scapula due to serratus ventralis tenotomy performed to remove the caudal insertion of the splenius capitis muscle. All the dogs recovered fully within one month, experiencing good clinical function. Histopathology showed complete tumour removal with no neoplastic fascial disruption in all cases. Compartmental excision provides effective local tumour control, representing an alternative to limb amputation or more radical excision if adjuvant radiotherapy is not an option for owners.

Background: Alzheimer's disease (AD) is clinically heterogeneous, including the classical-amnesic (CA-) phenotype and some variants., Objective: We aim to describe a further presentation we (re)named confabulation-misidentification (CM-) phenotype., Methods: We performed a retrospective longitudinal case-series study of 17 AD outpatients with the possible CM-phenotype (CM-ADs). Then, in a cross-sectional study, we compared the CM-ADs to a sample of 30 AD patients with the CA-phenotype (CA-ADs). The primary outcome was the frequency of cognitive and behavioral features. Data were analyzed as differences in percentage by non-parametric Chi Square and mean differences by parametric T-test., Results: Anterograde amnesia (100%) with early confabulation (88.2%), disorientation (88.2%) and non-infrequently retrograde amnesia (64.7%) associated with reduced insight (88.2%), moderate prefrontal executive impairment (94.1%) and attention deficits (82.3%) dominated the CM-phenotype. Neuropsychiatric features with striking misidentification (52.9%), other less-structured delusions (70.6%), and brief hallucinations (64.7%) were present. Marked behavioral disturbances were present early in some patients and very common at later stages. At the baseline, the CM-ADs showed more confabulation (p < 0.001), temporal disorientation (p < 0.02), misidentification (p = 0.013), other delusions (p = 0.002), and logorrhea (p = 0.004) than the CA-ADs. In addition, more social disinhibition (p = 0.018), reduction of insight (p = 0.029), and hallucination (p = 0.03) persisted at 12 months from baseline. Both the CA- and CM-ADs showed anterior and medial temporal atrophy. Compared to HCs, the CM-ADs showed more right fronto-insular atrophy, while the CA-ADs showed more dorsal parietal, precuneus, and right parietal atrophy., Conclusion: We described an AD phenotype resembling diencephalic rather than hippocampal amnesia and overlapping the past-century description of presbyophrenia.

Canine apocrine gland anal sac adenocarcinoma (AGASACA) is an aggressive canine tumor originating from the anal sac glands. Surgical resection, with or without adjuvant chemotherapy, represents the standard of care for this tumor, but the outcome is generally poor, particularly for tumors diagnosed at an advanced stage. For this reason, novel treatment options are warranted, and a few recent reports have suggested the activation of the immune checkpoint axis in canine AGASACA. In our study, we developed canine-specific monoclonal antibodies targeting PD-1 and PD-L1. A total of 41 AGASACAs with complete clinical and follow-up information were then analyzed by immunohistochemistry for the expression of the two checkpoint molecules (PD-L1 and PD-1) and the presence of tumor-infiltrating lymphocytes (CD3 and CD20), which were evaluated within the tumor bulk (intratumor) and in the surrounding stroma (peritumor). Seventeen AGASACAs (42%) expressed PD-L1 in a range between 5% and 95%. The intratumor lymphocytes were predominantly CD3+ T-cells and were positively correlated with the number of PD-1+ intratumor lymphocytes ( ρ = 0.36; p = 0.02). The peritumor lymphocytes were a mixture of CD3+ and CD20+ cells with variable PD-1 expression (range 0-50%). PD-L1 expression negatively affected survival only in the subgroup of dogs treated with surgery alone ( n = 14; 576 vs. 235 days). The presence of a heterogeneous lymphocytic infiltrate and the expression of PD-1 and PD-L1 molecules support the relevance of the immune microenvironment in canine AGASACAs and the potential value of immune checkpoints as promising therapeutic targets.

Osteosarcoma (OSA) is the most common pediatric malignant bone tumor. Although surgery together with neoadjuvant/adjuvant chemotherapy has improved survival for localized OSA, most patients develop recurrent/metastatic disease with a dismally poor outcome. Therapeutic options have not improved for these OSA patients in recent decades. As OSA is a rare and "orphan" tumor, with no distinct targetable driver antigens, the development of new efficient therapies is still an unmet and challenging clinical need. Appropriate animal models are therefore critical for advancement in the field. Despite the undoubted relevance of pre-clinical mouse models in cancer research, they present some intrinsic limitations that may be responsible for the low translational success of novel therapies from the pre-clinical setting to the clinic. From this context emerges the concept of comparative oncology, which has spurred the study of pet dogs as a uniquely valuable model of spontaneous OSA that develops in an immune-competent system with high biological and clinical similarities to corresponding human tumors, including in its metastatic behavior and resistance to conventional therapies. For these reasons, the translational power of studies conducted on OSA-bearing dogs has seen increasing recognition. The most recent and relevant veterinary investigations of novel combinatorial approaches, with a focus on immune-based strategies, that can most likely benefit both canine and human OSA patients have been summarized in this commentary.

Background: Hepatocellular carcinoma (HCC) in dogs is uncommon and often associated with a good prognosis, although some cases prove to be aggressive. In human oncology HCC is often very aggressive and diagnostic methods and prognostic factors are widely used to predict its biological behaviour. These include the expression of PIVKA-II., Methods: in order to identify a prognostic factor for canine HCC, we applied different methods of histological grading and investigated PIVKA-II expression in 22 HCC of dogs treated surgically and followed clinically for at least 2 years., Results: Nineteen patients analysed have passed the observation period without tumour recurrence, while 3 died following the development of metastases. PIVKA-II was positive in 15/22 cases without correlation with prognosis or tumoural grading even if a trend of PIVKA-II negativity in low WHO grades as well as increased number of PIVKA-II positive cases in higher WHO grades weres observed., Conclusions: This work showed that, PIVKA-II cannot be considered either as a marker of malignancy or as a prognostic marker for canine HCC. The poor prognosis depends usually on the clinical presentation. Thus prognostic parameters in canine HCC able to predict its aggressive behaviour through histological examination are still missing. The most promising method, limited to our study, seems to be the WHO histological grading.

Osteosarcoma is the most common primary malignant bone tumour in dogs, characterized by a locally aggressive and highly metastatic behaviour. Despite the current standards of care, most dogs succumb to the disease, indicating the need for novel treatment strategies. Polo-like kinase 1 (PLK1) is dysregulated in a variety of human cancer types, including osteosarcoma, and induces c-Myc accumulation. The crosstalk between the two molecules coordinates cell proliferation, differentiation, self-renewal and apoptosis. Therefore, PLK1 has recently emerged as a potential therapeutic target, mainly in tumours overexpressing c-Myc. BI 2536 is a selective PLK1 inhibitor promoting mitotic arrest and apoptosis in a variety of cancer cells. This research aimed at evaluating PLK1 and c-Myc protein expression in 53 appendicular canine osteosarcoma (cOSA) samples and the in vitro effects of BI 2536 on a c-Myc and PLK1-overexpressing cOSA cell line (D17). PLK1 and c-Myc expression in cOSA samples showed no correlation with clinicopathological data. However, c-Myc overexpression was associated with a significantly reduced overall survival (p = .003). Western Blot and RT-qPCR assays revealed that D17 expressed high protein and transcript levels of both PLK1 and MYC. When treated with BI 2536 (range 2.5-15 nM) for 24 h, D17 showed a substantial decrease in cell growth, inducing apoptosis and G 2 /M cell cycle arrest. Interestingly, under BI 2536 treatment, D17 showed decreased c-Myc protein levels. Consistent with human OSA, these preliminary data outline the prognostic value of c-Myc expression in cOSA and highlight the potential role of PLK1 as an antiproliferative therapeutic target for tumours overexpressing c-Myc., (© 2022 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

The development of chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematological diseases. However, approximately 60% of patients relapse after CAR-T cell therapy, and no clear cause for this failure has been identified. The objective of the Bio-CAR-T BS study (ClinicalTrials.gov: NCT05366569) is to improve our understanding of the lymphocyte harvest to maximize the quality of the CAR-T cell product. Of the 14 patients enrolled, 11 were diagnosed with DLBCL, 2 with PMBCL, and 1 with ALL. Five of 11 DLBCL patients met the criteria for "pre-emptive" Lymphocytes-apheresis (being at high risk of second relapse), and 6 were included in the standard-of-care Lymphocytes-apheresis group. Previous autologous stem cell transplantation (ASCT) and age were significantly different between the two groups. At the time of Lymphocyte-apheresis, patients in the "pre-emptive" group had more "fit" lymphocytes (higher CD4+/CD8+ ratio; higher naïve T cells levels) compared with standard group, probably due to the impact of ASCT. At the same time, also being older than 60 years results in a more "exhausted" lymphocyte profile. Overall, "pre-emptive" Ly-apheresis in DLBCL patients at high risk of relapse appears to be feasible and may allow the timely collection of "fit" lymphocytes for CAR-T cell manufacturing.

Regulatory small RNAs (sRNAs) are involved in the adaptation of bacteria to their environment. CiaR-dependent sRNAs (csRNAs) are controlled by the regulatory two-component system (TCS) CiaRH, which is widely conserved in streptococci. Except for Streptococcus pneumoniae and Streptococcus sanguinis, the targets of these csRNAs have not yet been investigated. Streptococcus agalactiae, the leading cause of neonatal infections, has four conserved csRNA genes, namely, srn015 , srn024 , srn070 , and srn085 . Here, we demonstrate the importance of the direct repeat TTTAAG-N5-TTTAAG in the regulation of these csRNAs by CiaRH. A 24-nucleotide Srn024- sap RNA base-pairing region is predicted in silico . The sap gene encodes a LPXTG-cell wall-anchored pullulanase. This protein cleaves α-glucan polysaccharides such as pullulan and glycogen present in the environment to release glucose and is involved in adhesion to human cervical epithelial cells. Inactivation of S. agalactiae pullulanase (SAP) leads to no bacterial growth in a medium with only pullulan as a carbon source and reduced biofilm formation, while deletion of ciaRH and srn024 genes significantly increases bacterial growth and biofilm formation. Using a new translational fusion vector, we demonstrated that Srn024 is involved in the posttranscriptional regulation of sap expression. Complementary base pair exchanges in S. agalactiae suggest that Srn024 interacts directly with sap mRNA and that disruption of this RNA pairing is sufficient to yield the biofilm phenotype of Srn024 deletion. These results suggest the involvement of Srn024 in the adaptation of S. agalactiae to environmental changes and biofilm formation, likely through the regulation of the sap gene. IMPORTANCE Although Streptococcus agalactiae is a commensal bacterium of the human digestive and genitourinary tracts, it is also an opportunistic pathogen for humans and other animals. As the main cause of neonatal infections, it is responsible for pneumonia, bacteremia, and meningitis. However, its adaptation to these different ecological niches is not fully understood. Bacterial regulatory networks are involved in this adaptation, and the regulatory TCSs (e.g., CiaRH), as well as the regulatory sRNAs, are part of it. This study is the first step to understand the role of csRNAs in the adaptation of S. agalactiae. This bacterium does not currently exhibit extensive antibiotic resistance. However, it is crucial to find alternatives before multidrug resistance emerges. Therefore, we propose that drugs targeting regulatory RNAs with Srn024-like activities would affect pathogens by reducing their abilities to form biofilm and to adapt to host niches.

Exosomes are extracellular vesicles playing a pivotal role in the intercellular communication. They shuttle different cargoes, including nucleic acids from their cell of origin. For this reason, they have been studied as carriers of tumor markers in different liquid biopsy approaches, in particular for solid tumors. Few data are available concerning exosomes as markers of myeloid neoplasia. To better understand their real potential and the best approach to investigate leukemic exosomes, we present the results of a pilot feasibility study evaluating the application of next-generation sequencing analysis of dsDNA derived from exosomes isolated in 14 adult patients affected by acute myeloid leukemias. In particular, leukemia-derived exosome fractions have been analyzed. The concentration of dsDNA co-extracted with exosomes and the number and types of mutations detected were considered and compared with ones identified in the Bone Marrow (BM) and Peripheral Blood (PB) cells. Exosomal DNA concentration, both considering the cargo and the DNA surrounding the lipid membrane resulted in a linear correlation with leukemic burden. Moreover, exosomal DNA mutation status presented 86.5% of homology with BM and 75% with PB. The results of this pilot study confirmed the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA NGS analysis for AML biomarker detection. These data point to the use of liquid biopsy in myeloid neoplasia for the detection of active leukemic cells resident in the BM via a painless procedure.

Malnutrition is common after allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT), and interventions directed to correct nutritional status are warranted to improve transplant outcomes. In this prospective study, an oral polymeric formulation enriched with TGF-β2 (TE-OPF) was explored to correct malnutrition according to Patient-Generated Subjective Global Assessment (PG-SGA). TE-OPF was proposed to 51 consecutive patients who received transplants at our institution for hematological malignancies, and sufficient dose intake was established per protocol as at least 50% of the prescribed dose of TE-OPF: group A received adequate nutritional support; group B, inadequate. The study met the primary outcomes in terms of safety (no adverse events reported during TE-OPF intake except for its disgusting taste) and malnutrition (PG-SGA C 28 days after transplant): severely malnourished patients (PG-SGA C) accounted for 13% in group A and 88.9% in group B ( p = 0.000). At the end of the study, after a median follow-up of 416 days, the estimated median Overall Survival (OS) was 734 days for well or moderately nourished patients (PG-SGA A/B) in comparison to 424 for malnourished patients ( p = 0.03). Inadequate TE-OPF intake was associated with an increase in acute gastrointestinal Graft Versus Host Disease (GVHD) cumulative incidence (38% vs. 0% p = 0.006). A higher incidence of pneumonia was reported in group B ( p = 0.006). IGF-1 levels at 14 and 28 days after transplant were significantly higher in group A and were associated with a lower incidence of acute GVHD (aGVHD). Higher subsets of B, T, and NK cells were found in group A, and a higher number of CD16+ NK cells was associated with a lower incidence of acute GVHD ( p = 0.005) and increased survival at the end of the study ( p = 0.023). Artificial neural network analysis suggested that inadequate TE-OPF intake, pneumonia, and sepsis significantly affected malnutrition 28 days after alloHSCT and survival 365 days after alloHSCT (normalized importance 100%, 82%, and 68%, respectively). In this exploratory and preliminary study, the use of TE-OPF appeared to reduce the incidence of malnutrition after alloHSCT, but larger and controlled studies are required.

Background: Infectious complications are a significant cause of morbidity and mortality in patients undergoing allogeneic haematopoietic stem cell transplantation (Allo-SCT). The BATMO (Best-Antimicrobial-Therapy-TMO) is an innovative program for infection prevention and management and has been used in our centre since 2019. The specific features of the BATMO protocol regard both prophylaxis during neutropenia (abandonment of fluoroquinolone, posaconazole use in high-risk patients, aerosolized liposomal amphotericin B use until engraftment or a need for antifungal treatment, and letermovir use in CMV-positive recipients from day 0 to day +100) and therapy (empirical antibiotics based on patient clinical history and colonization, new antibiotics used in second-line according to antibiogram with the exception of carbapenemase-producing K pneumoniae for which the use in first-line therapy is chosen)., Methods: Data on the infectious complications of 116 transplant patients before BATMO protocol (Cohort A; 2016 - 2018) were compared to those of 84 transplant patients following the introduction of the BATMO protocol (Cohort B; 2019 - 2021). The clinical and transplant characteristics of the 2 Cohorts were comparable, even though patients in Cohort B were at a higher risk of developing bacterial, fungal, and CMV infections, due to a significantly higher proportion of myeloablative regimens and haploidentical donors., Results: No change in the incidence of infections with organ localization was observed between the two Cohorts. A significant reduction in Clostridioides difficile infections by day +100 was observed in Cohort B (47% vs. 15%; p=0.04). At day +30, a higher incidence of Gram-negative bloodstream infections (BSIs) was observed in Cohort B (12% vs. 23%; p=0.05). By day +100 and between days +100 and +180, the incidence of BSIs and of the various etiological agents, the mortality from Gram-negative bacteria, and the incidence of invasive fungal infections were not different in the two Cohorts. The incidence of CMV reactivations by day +100 dropped drastically in patients of Cohort B, following letermovir registration (51% vs. 15%; p=0.00001)., Discussion: The results of this study suggest that the BATMO program is safe. In particular, the choice to avoid prophylaxis with fluoroquinolone was associated with an increase in Gram-negative BSIs by day +30, but this did not translate into higher levels of mortality. Moreover, this strategy was associated with a significant reduction of Clostridiodes difficile infections. The efficacy of anti-CMV prophylaxis with letermovir was confirmed by a significant reduction in CMV reactivations. Even though patients in Cohort B were at higher risk of developing fungal infections (more haploidentical transplants with more myeloablative regimens), the extensive use of posaconazole for prophylaxis balanced this risk, and no increase in the incidence of fungal-associated complications was observed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Malagola, Turra, Signorini, Corbellini, Polverelli, Masina, Del Fabro, Lorenzotti, Fumarola, Farina, Morello, Radici, Buttini, Colnaghi, Bernardi, Re, Caruso, Castelli and Russo.)

Background: Melanoma is the most lethal form of skin cancer in humans. Conventional therapies have limited efficacy, and overall response is still unsatisfactory considering that immune checkpoint inhibitors induce lasting clinical responses only in a low percentage of patients. This has prompted us to develop a vaccination strategy employing the tumor antigen chondroitin sulfate proteoglycan (CSPG)4 as a target., Methods: To overcome the host's unresponsiveness to the self-antigen CSPG4, we have taken advantage of the conservation of CSPG4 sequence through phylogenetic evolution, so we have used a vaccine, based on a chimeric DNA molecule encompassing both human (Hu) and dog (Do) portions of CSPG4 (HuDo-CSPG4). We have tested its safety and immunogenicity (primary objectives), along with its therapeutic efficacy (secondary outcome), in a prospective, non-randomized, veterinary clinical trial enrolling 80 client-owned dogs with surgically resected, CSPG4-positive, stage II-IV oral melanoma., Results: Vaccinated dogs developed anti-Do-CSPG4 and Hu-CSPG4 immune response. Interestingly, the antibody titer in vaccinated dogs was significantly associated with the overall survival. Our data suggest that there may be a contribution of the HuDo-CSPG4 vaccination to the improvement of survival of vaccinated dogs as compared with controls treated with conventional therapies alone., Conclusions: HuDo-CSPG4 adjuvant vaccination was safe and immunogenic in dogs with oral melanoma, with potential beneficial effects on the course of the disease. Thanks to the power of naturally occurring canine tumors as predictive models for cancer immunotherapy response, these data may represent a basis for the translation of this approach to the treatment of human patients with CSPG4-positive melanoma subtypes., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)