Immune adjuvants are immune modulators that have been developed in the context of infectious vaccinations. There is currently a growing interest in immune adjuvants due to the development of immunotherapy against cancers. Immune adjuvant mechanisms of action are focused on the initiation and amplification of the inflammatory response leading to the innate immune response, followed by the adaptive immune response. The main activity lies in the support of antigen presentation and the maturation and functions of dendritic cells. Most immune adjuvants are associated with a vaccine or incorporated into the new generation of mRNA vaccines. Few immune adjuvants are used as drugs. Hydroxyapatite (HA) ceramics and azoximer bromide (AZB) are overlooked molecules that were used in early clinical trials, which demonstrated clinical efficacy and excellent tolerance profiles. HA combined in an autologous vaccine was previously developed in the veterinary field for use in canine spontaneous lymphomas. AZB, an original immune modulator derived from a class of heterochain aliphatic polyamines that is licensed in Russia, the Commonwealth of Independent States, and Slovakia for infectious and inflammatory diseases, is and now being developed for use in cancer with promising results. These two immune adjuvants can be combined in various immunotherapy strategies.

Background Pet dogs spontaneously develop lymphoma. An anthracycline-based multidrug chemotherapy regimen represents the treatment cornerstone; however, cure is rarely achieved. We have been treating dogs with B-cell lymphoma with an autologous vaccine (APAVAC®) and CHOP-based chemotherapy since 2011. Methods To better characterize the safety and efficacy of APAVAC®, and to find the best candidates for immunotherapy, we designed a retrospective study on all dogs treated with chemo-immunotherapy to date and compared them with those dogs treated with chemotherapy only. All dogs were completely staged and re-staged at the end of treatment. The primary endpoint was the effectiveness of chemo-immunotherapy, measured as time to progression (TTP), lymphoma-specific survival (LSS), and 1-, 2-, and 3-year survival rates. The secondary objective was safety. Results Three hundred dogs were included: 148 (49.3%) received chemotherapy and 152 (50.7%) chemo-immunotherapy. Overall, the latter survived significantly longer (median LSS, 401 vs 220; P < 0.001). Among dogs with diffuse large B-cell lymphoma, the 1-, 2- and 3-year survival rates were 20, 13 and 8% for chemotherapy, and 51, 19 and 10% for chemo-immunotherapy. The benefit of chemo-immunotherapy was particularly relevant in dogs with concurrent high serum LDH, stage V, substage a disease and not previously treated with steroids (median LSS, 480 vs 85 days; P < 0.001). Among dogs with nodal marginal zone lymphoma, those having at least 3 of the aforementioned characteristics significantly benefited from chemo-immunotherapy (median LSS, 680 vs 160 days, P < 0.001). The 1-, 2- and 3-year survival rates were 30, 16 and 10% for chemotherapy, and 55, 28 and 10% for chemo-immunotherapy. Among dogs with follicular lymphoma, lack of immunotherapy administration was the only variable significantly associated with increased risk of tumor-related death. Chemo-immunotherapy was remarkably well tolerated, with no local or systemic adverse events. Conclusions Overall, the addition of immunotherapy to a traditional CHOP protocol is associated with improved outcome in dogs with B-cell lymphoma, regardless of histotype and evaluated prognostic factors. Moreover, the identikit of the best candidate for immune-therapy was delineated for the most common histotypes. The study also confirms the excellent tolerability of the vaccine.

Bone function requires a good repair system. The quantitative role of each healing component, mainly periosteum and bone marrow/endosteum and of their interactions is not clearly known. To evaluate more precisely the role of periosseous tissues in bone formation, we chose a bone lengthening model in rabbits. Nineteen purebred immature male New Zealand white rabbits were fitted with a femoral lateral external fixator and randomly assigned to 4 intervention groups: 1) Periosteum (PS) and bone marrow (BM) preservation; 2) PS preservation and BM destruction; 3) PS destruction and BM preservation; 4) PS and BM destruction. Animals were killed on day 30 and histology showed different bone regeneration modes. The most frequent ossification in the centre of the distraction gap was enchondral ossification. For bone formed between cortices only group 1 showed a difference (p=0.034). Density of the formed bone at the periosteal site showed a positive effect only with PS preservation, with an interaction of BM and PS. Density of group 4 was different from groups 1 (p=0.028), and 2 (p=0.001). No difference was present between groups at the endosteal sites. PS had a positive effect on the thickness of the formed bone at the periosteal site, with an interaction between PS and BM, but not at the endosteal site. A difference in thickness was noted between group 2 and 4 (p=0.018). Periosteum forms more bone and more likely to spread than bone marrow, but for restoration of the cortical wall, preservation of both shows optimal density results. Bone does not seem to form from surrounding tissues when both periosteum and bone marrow are destroyed.

Abstract Introduction. Nowadays, immune therapy is a major therapy for C. Vaccination was developed for more than 20 years (y) with less than 20% of responses (R) associated with a mild advantage in overall survival (OS) in addition to its complexity. APAVAC is an AV consisting of hydroxyapatite ceramic powder (HA) and specific tumor proteins purified from the C or from the serum (S). HA is aimed to purify and vectorize C proteins containing heat shock proteins and C antigens (Ag). So, AV associates an adjuvant, HA, and autologous C Ag. A randomized, placebo-controlled, double-blind chemo-APAVAC clinical trial in pet dog model of diffuse large B-cell lymphoma (L) showed significant OS for the APAVAC arm (Marconato L et al. Clin Cancer Res 2013). Patients. 67 pts (30 males and 37 females) were included in this pilot study. All of these pts had advanced disease (D), in relapse or in refractory relapse. AV was weekly administered by sc for 1 mo followed by monthly injection for at least 4 mo. Pts received at least 1 AV. Evaluation included R rate; OS was calculated from the beginning of the AV. Results.The median age was 65y (range:14-85). 31 pts received an incomplete cycle of AV. 16 of them have ongoing therapy. Among the whole population, 14 pts died with 5 due to associated therapy and 9 early deaths directly due to the D. 36 pts received a complete cycle of AV (at least 8 injections) with follow-up >6 months (mo). Tolerance was excellent, with only mild local inflammation or pain, controlled by lidocaine patch. No auto-immune manifestations were observed. Among the 36 pts having a complete cycle of AV, 11 had AV from the C and 25 from the S. 33 of them had solid C, including 10 glioblastoma grade IV with 8 having 3 previous lines of therapy (PLT) and 2 having 1 PLT; 6 had Progressive D (PD) and 4, Stable D (SD) at the inclusion followed by 5 SD and 5 PD, all of them with a median OS of 12 mo; 1 bladder C with 1 PLT in PR, followed by CR lasting 22mo+; 2 endometrial C including 1 with metastatic (M) D, having 1 PLT in PD followed by 1 SD and 1 PR lasting 69 mo+ and 30 mo+; 1 M prostatic C, having 1 PLT in PD followed by SD lasting 58 mo+; 3 head and neck C with 1 M having 1 PLT, all with PD followed by PR lasting 56 mo+, 12 mo+ and 6 mo+; 3 renal cell C including 2 M with 1 PLT in PD followed by 2 PR and 1 PD lasting 16 mo+ for 2 pts and 10 mo for 1pt; 2 liver C with 2 PLT, both in PD followed by PR lasting 18 mo+; 2 peritoneal C with 2 PLT in PD followed by 1 PD and 1 CR lasting 6 mo and 6 mo+; 3 M colon C, 1 with 1 PLT and 2 with 2 PLT, all in PD followed by 1 PR and 2 CR lasting 6 mo, 24 mo+ and 18 mo+; 2 gastric C with 1 and 2 PLT, all in PD followed by 1 PD and 1 PR lasting 6 mo and 6 mo+; 2 sarcomas including 1 Ewing, all with 2 PLT in PD followed by 1 PD and 1 PR lasting 9 mo and 9 mo+; 1 lung cancer with 2 PLT in PD followed by PD at 12 mo+; 1 M melanoma with 2 PLT in PD followed by PR at 6 mo+. 3 pts had HM including: 2 mantle cell lymphomas (MCL) with 3 PLT for 1 pt and 7 PLT for the other, all in PR after ibrutinib followed by CR under AV alone for 1 pt lasting 12 mo+. The second pt had leukemic C cells decreasing at 2 mo of AV alone; 1 transformed Waldenström's disease (WD) having 5 PLT, in PR after venotoclax followed by AV alone, with only IgM level at 4 g/L and no tumor, at 6 mo+. 9 pts were controlled with AV alone for 12 mo+. In 1 pt analyzed, 2 T-cell receptor (TCR) clonal rearrangements were observed in peripheral blood after 3 mo. 8 additional pts with HM had ongoing therapy and will be presented. 1 pt had GIST and follicular (F) L, 1 had WD, 1 had MCL, 3 had multiple myeloma with 1 responding at 4 injections, 1 pt with osteomyelofibrosis and 1 with myelodysplastic syndrome. Conclusions. APAVAC is a simple method for AV with high clinical impact in very bad situations. It could be used in combination with other therapy and for residual disease control. Further clinical studies are planned in solid C as well as in HM. Disclosures Rouquet: URODELIA: Employment. Frayssinet:URODELIA: Employment.

Calcium phosphate coprecipitated with plasmid DNA (pDNA) has been used for more than two decades for in vitro cell transfection. We have evaluated calcium phosphate ceramic powder to transfect cells in vitro and in vivo . It appeared that the transfection yield was dependent on several factors such as the amount of powder in contact with the cells, and the time of contact. In vivo , cells that were in the vicinity of the ceramic particles were the first to be transfected – in particular those involved in the foreign body reaction occurring at the contact of the particles.

The aim of this non-randomized controlled trial was to compare time to progression (TTP), lymphoma-specific survival (LSS), and safety of an autologous vaccine (consisting of hydroxyapatite ceramic powder and Heat Shock Proteins purified from the dogs' tumors, HSPPCs-HA) plus chemotherapy versus chemotherapy alone in dogs with newly diagnosed, clinically advanced, histologically confirmed, multicentric indolent B-cell lymphoma. The vaccine was prepared from dogs' resected lymph nodes and administered as an intradermal injection. Forty-five client-owned dogs were enrolled: 20 dogs were treated with dose-intense chemotherapy, and 25 received concurrent immunotherapy. Both treatment arms were well tolerated, with no exacerbated toxicity in dogs also receiving the vaccine. TTP was significantly longer for dogs treated with chemo-immunotherapy versus those receiving chemotherapy only (median, 209 versus 85 days, respectively, P=0.015). LSS was not significantly different between groups: dogs treated with chemo-immunotherapy had a median survival of 349 days, and those treated with chemotherapy only had a median survival of 200 days (P=0.173). Among vaccinated dogs, those mounting an immune response had a significantly longer TTP and LSS than those with no detectable response (P=0.012 and P=0.003, respectively). Collectively these results demonstrate that vaccination with HSPPCs-HA may produce clinical benefits with no increased toxicity, thereby providing a strategy for enhancing chemotherapy in dogs with advanced indolent lymphoma.

An autovaccine against canine DLBCL lymphomas associated to a CHOP protocol has proven to greatly improve the overall survival and the time to progression of a series of dogs against another series only treated by the CHOP protocol (1). This vaccine was obtained by purifying specific proteins and peptides from a node biopsy. In order that the vaccine matches the heterogeneity of the tumor, one vaccine was produced for each dog.

The most promising techniques for detecting minimal residual disease (MRD) in canine lymphoma are flow cytometry (FC) and polymerase chain reaction amplification of antigen receptor genes (PARR). However, the agreement between these methods has not been established. MRD was monitored by FC and PARR following treatment of dogs affected with diffuse large B-cell lymphoma (DLBCL), comparing results in lymph node (LN), peripheral blood (PB) and bone marrow (BM) samples. The prognostic impact of MRD on time to relapse (TTR) and lymphoma-specific survival (LSS) was also assessed. Fourteen dogs with previously untreated DLBCL were enrolled into the study; 10 dogs eventually relapsed, while four dogs with undetectable MRD were still in remission at the end of the study. At diagnosis, the concordance rate between FC and PARR was 100%, 78.6%, and 64.3% for LN, PB and BM, respectively. At the end of treatment, the agreement rates were 35.7%, 50%, and 57.1% for LN, PB and BM, respectively. At least one of the follow-up samples from dogs experiencing relapse was PARR+; conversely, FC was not able to detect MRD in seven of the dogs that relapsed. PARR was more sensitive than FC in predicting TTR, whereas the combination of PARR and FC was more sensitive than either technique alone in predicting LSS using PB samples. The results suggest that immunological and molecular techniques should be used in combination when monitoring for MRD in canine DLBCL.

Abstract Purpose: Active immunotherapy is a promising antitumoral strategy; however its use in combination with chemotherapy in dogs with large B-cell lymphoma (DLBCL) remains largely untested. Heat shock proteins (HSP) bind the small peptides they chaperone (HSPPC), allowing for immunization of the host against a large repertoire of tumor-associated antigens. Hydroxylapatite vehicles HSPPCs and acts as an immunologic adjuvant. The aim of this study was to show that an autologous vaccine with hydroxylapatite and tumor-derived HSPPCs is safe and therapeutically effective in dogs with DLBCL. Experimental Design: Nineteen dogs with naturally occurring DLBCL were entered into a prospective randomized placebo-controlled double-blinded trial of HSPPCs–hydroxylapatite plus chemotherapy versus chemotherapy alone. Endpoints included time to progression (TTP), lymphoma-specific survival (LSS), and incidence of toxicoses. Results: Median first TTP after randomization to the vaccine arm was 304 days versus 41 days for the control arm (P = 0.0004). There was also a statistically significant difference in duration of second remission between the two groups (P = 0.02). Median LSS was 505 days for the vaccinated dogs versus 159 days for the unvaccinated dogs (P = 0.0018). Six vaccinated dogs achieved molecular remission, as shown by clonal immunoglobulin H (IgH) rearrangement. Toxicoses were comparable between the two treatment arms. Conclusions: The results of this trial demonstrate that the autologous vaccine tested here is safe and efficacious in prolonging TTP and LSS in dogs with DLBCL when used in combination with dose-intense chemotherapy. On the basis of these results, additional evaluation of this novel therapeutic strategy is warranted in human DLBCL. Clin Cancer Res; 20(3); 668–77. ©2013 AACR.

Sixty-three dogs with newly diagnosed lymphoma underwent complete staging and received the same chemotherapy. Diffuse large B-cell lymphoma was the leading histotype (44.4%), followed by peripheral T-cell lymphoma (20.6%). Indolent lymphomas accounted for 30.2% of cases. Most dogs with aggressive B-cell lymphoma had stage IV disease. Dogs with indolent and aggressive T-cell lymphoma had more often stage V disease and were symptomatic. Liver and bone marrow were predominantly involved in B-cell and T-cell lymphoma, respectively. The clinical stage was significantly related to substage, sex and total lactic dehydrogenase (LDH) levels. Aggressive B-cell lymphomas were more likely to achieve remission. Median survival was 55 days for aggressive and indolent T-cell lymphoma, 200 and 256 days for indolent and aggressive B-cell lymphoma, respectively. The prognosis of advanced indolent lymphoma does not appear to be appreciably different from that of aggressive disease. Familiarity with the various histotypes is critical to make the correct diagnosis and drive therapy.

Randomized, placebo-controlled, double-blinded chemo1 immunotherapy clinical trial in a Pet Dog model of Diffuse Large B2 cell Lymphoma 3 4 Laura Marconato , Patrick Frayssinet , Nicole Rouquet , Stefano Comazzi , Vito 5 Ferdinando Leone , Paola Laganga , Federica Rossi , Massimo Vignoli , Lorenzo 6 Pezzoli , Luca Aresu 5 7 8 9 Authors’ affiliations: 1 Centro Oncologico Veterinario, Sasso Marconi, Bologna, Italy; 2 10 Urodelia, St Lys, France; 3 Department of Veterinary Sciences and Public Health, 11 University of Milan, Italy; 4 Epidemiology advisor, London, UK; 5 Department of 12 Comparative Biomedicine and Food Science, University of Padova, Italy. 13 14 15 Running title: Chemo-immunotherapy for canine DLBCL 16 17 18 Key-words: diffuse large B-cell lymphoma, dog, active immunotherapy, heat shock 19 proteins, hydroxylapatite 20 21 22 *Corresponding author: 23 Laura Marconato, Centro Oncologico Veterinario, via San Lorenzo 1-4, 40037 Sasso 24 Marconi (BO), Italy; Ph/Fax: 0039 051 6751871; e-mail: 25 marconato@centroncologicovet.it 26 27 28 Conflicts of interest: Two authors (P.F., N.R.) are employed by a company 29 (URODELIA) whose product was studied in the present work. 30 31

Chemotherapy protocols have also been developed for dogs in the last 40 years. They have proved to be effective for the overall survival of the treated animal. Although the order of drug administration and duration of the maintenance part of the protocol vary considerably, most oncologists agree that a doxorubicin-based (eg CHOP) combination chemotherapy protocol provides the longest period of disease control and overall survival [3].

Magnetite nanoparticles are frequently used to eliminate by heating in a high frequence oscillating magnetic field the tumor cells into which they are introduced in order to directly kill the cells or to make them more sensitive to radiotherapy (Ito, A., et al., 2005; Jordan, A., et al., 2001). The appearance of bone metastases is a sign of a dissemination of primitive cancers. They rapidly become resistant to chemotherapy and radiotherapy and are often very painful necessitating local and/or alternative treatments in order to reduce the osteolysis triggered by the cancerous cells. The osteolysis is due to local activation of the osteoclasts and macrophages by factors synthesized by the tumor cells (Shimamura, T., et al., 2005). It is the osteolysis that is very often responsible for the pain. We have developed a biomaterial containing magnetite nanoparticles which can be introduced into bone metastases in order to release naked nanoparticles in the contact with both the cancerous and the osteolytic cells. The material is made of a calcium sulphate paste containing a small percentage of nanoparticles which can be injected inside the metastasis (fig. 1). It sets within a few minutes in situ. The degradation of the calcium sulphate matrix within a few days releases the nanoparticles which are then available for cell internalisation. In vitro, these particles can be internalised in high amounts by metastatic cells from adenocarcinoma. The number of nanoparticles found inside the cells depends on the nanoparticle size, however the mass internalized seems to be almost independent of their size (Frayssinet, P., et al., 2005). The nanoparticles did not show in vitro any signs of cell toxicity. This is consistent with previous reports which showed that cytotoxicity of magnetite nanoparticles could be due to several factors such as coating (Hafeli & Pauer, 1999). Furthermore, they are intented for use in very low doses (a few mgs). The degradation products of iron oxide are well known. They do not have a reported toxicity and are easily eliminated from the organism (Schoepf, U., et al. 1998, Okon, E.E., et al. 2000, Okon, F., et al. 1994). Migration of the nanoparticles can however be a cause of concern due to the possible unwanted heating of other regions of the organism when submitted to a magnetic field.

The proinflammatory and catabolic cytokine IL-1β has been implicated in the pathogenesis of osteoarthritis (OA) by mediating synovial inflammation and cartilage degeneration. Although synovial macrophages are suggested to be the source of IL-1β, the mechanism remains unclear. Ectopic deposition of hydroxyapatite (HA) crystals in joints is closely associated with OA and other arthropathies, but the precise role of HA in arthritis pathogenesis has not been clearly demonstrated. Here we show that HA crystals of a particular size and shape can stimulate robust secretion of proinflammatory cytokines IL-1β and IL-18 from murine macrophages in a NLRP3 inflammasome-dependent manner. HA-induced inflammasome activation is dependent on potassium efflux, generation of reactive oxygen species (ROS), and lysosomal damage, but independent of cell death. Mice lacking the inflammasome components are protected against HA-induced neutrophilic inflammation in the air-pouch model of synovitis, and they show decreased joint pathology accompanying spontaneous HA deposition in the ank-deficient mouse model of arthritis. Moreover, calcium crystal positive synovial fluids from some OA patients exhibited inflammasome-stimulatory activity in vitro. These results demonstrate that the NLRP3 inflammasome mediates the pathological effect of HA crystals in vitro and in vivo and suggest a critical role for the inflammasome in the pathogenesis of OA.

Introduction: Angular deformity in the growing skeleton of animals, especially in the radius and ulna, is occasionally seen in clinical practice. The mechanism of spontaneous correction of these angular deformities however remains to be elucidated. The purpose of our experiment was to explore the ability of a growth plate to correct an induced valgus deformity; and to study the mechanism of correction.Methods: Before beginning the study, valgus deformity of the distal radius had been induced in lambs by the application of a device that causes asymmetrical compression of the growth plate. The study began after removal of the device and spontaneous correction of the induced deformity was observed weekly for 20 weeks. The angles of the deformity and longitudinal growth on the medial and lateral portions of the growth plate were respectively measured on craniocaudal and mediolateral radiographs.Results and conclusions: Spontaneous correction of the valgus deformity occurred during the first 16 weeks. It resulted from asymmetrical growth characterised by restricted activity of the medial portion of the growth plate (14.8%) in comparison to the lateral portion of the experimental radius, and also in comparison to the medial portion of the control radius.