Gerardo Claudio Glikin

We present here the feasibility, safety, and efficacy results of a 7‐year study that included 337 canine melanoma patients undergoing surgery with adjuvant intralesional therapies combined with immunogenic therapy. After complete (CS) or partial (PS) surgery, the patients received post‐surgical bed injections of (i) lipoplexes carrying canine interferon‐β (cIFNβ) gene combined with bleomycin (CT _IF/B ) or (ii) 5‐fluorouracil (CT _5FU ). This surgery adjuvant therapy (SAT) also included the periodic administration of subcutaneous genetic vaccines composed of tumour extracts and lipoplexes carrying cIFNβ, human interleukin‐2, and human granulocyte‐macrophage colony‐stimulating factor genes. Compared at the end of their individual follow‐ups with controls treated with complete surgery alone (CSo), CS‐CT _IF/B and CS‐CT _5FU treatments quadrupled the fraction of local disease‐free (from 19% to 81% and 85%), and increased the percentage of metastasis‐free patients (M0: from 47% to 82% and 81%). Both PS arms (PS‐CT _IF/B and PS‐CT _5FU ) also significantly increased the fraction of metastatic‐free disease (M0: from 48% to 75% and 73%). In addition, SAT produced a significant 11‐ (CS‐CT _IF/B : p < 0.0001, HR _{95% CI} = 0.155 [0.109–0.222]), 9‐ (CS‐CT _5FU : p < 0.0001, HR _{95% CI} = 0.172 [0.123–0.242]), 6‐ (PS‐CT _IF/B : p < 0.0001, HR _{95% CI} = 0.204 [0.132–0.315]), and 7‐ (PS‐CT _5FU : p < 0.0001, HR _{95% CI} = 0.203 [0.132–0.311]) fold increase of overall survival as compared to their respective CSo and PSo controls. The respective median follow‐up days for each group were: 695.5 (28‐2516), 542.5 (46‐2554), 458 (29‐1273), and 521 (30‐1659). In general terms, this SAT transformed a lethal disease into a manageable condition where 25% (CS‐CT _IF/B ), 24% (CS‐CT _5FU ), 22% (PS‐CT _IF/B ), and 22% (PS‐CT _5FU ) of patients were still alive at the end of the study, while 53%, 60%, 16%, and 17%, respectively, died from causes unrelated to melanoma. Both surgery adjuvant treatments delayed or prevented post‐surgical recurrence and metastases, improved disease‐free and overall survival while maintaining quality of life. These successful outcomes encourage assaying a similar scheme for human melanoma.

Introduction: Melanoma is considered the deadliest form of skin cancer. While monoclonal-antibodies and molecular targets marked milestones in melanoma therapy, more research is needed to overcome the advanced stages of this disease. Objective: To explore the possible use of the yeast cytosine deaminase::uracil phosphoribosyltransferase fusion enzyme/5-fluorocytosine (CD::UPRT/5FC) suicide gene (SG) system for human melanoma. Methods: In eight metastatic human melanoma cell lines, we determined: cytotoxicity, lipofection efficiencies, colony forming capacity and bystander effects due to soluble and/or particulate factors secreted to the conditioned media after treatments. Results: CD::UPRT induced cell death in a prodrug (5FC) concentration-dependent manner and was able to eliminate the sub-population of surviving cells with clonogenic capacity. Compared with human interferon-β gene transfer or the herpes simplex virus thymidine kinase/ganciclovir system, at 100 μM 5FC, CD::UPRT was more efficient in inducing cell death. The strong cytotoxic response contrasted with the low lipofection efficiencies (<5%), indicating a potent bystander effect. We analyzed the contribution of soluble and particulate factors released by SG lipofected cells to the conditioned media (CM) finding that they were able to deliver CD::UPRT genetic information and/or recombinant enzyme to recipient cells. When exposed to 5FC, the cells that received either supernatant or 12000×g pellet fractions of CM, efficiently activated the prodrug because of the acquired CD::UPRT activity and caused cell death. Conclusion: This suicide gene therapy approach, amplified by the release of free 5-fluorouracil and soluble and particulate factors containing CD::UPRT genetic information and/or enzyme, could have a great clinical potential for malignant melanoma.

We tested the efficacy of a yeast cytosine deaminase::uracil phosphoribosyl transferase/5-fluorocytosine (CDU/5-FC) non-viral suicide system on eight established canine melanoma cell lines. Albeit with different degree of sensitivity five days after lipofection, this system was significantly efficient killing melanoma cells, being four cell lines highly, two fairly and two not very sensitive to CDU/5-FC (their respective IC₅₀ ranging from 0.20 to 800 μM 5-FC). Considering the relatively low lipofection efficiencies, a very strong bystander effect was verified in the eight cell lines: depending on the cell line, this effect accounted for most of the induced cell death (from 70 to 95 %). In our assay conditions, we did not find useful interactions either with the herpes simplex thymidine kinase/ganciclovir suicide system (in sequential or simultaneous modality) or with cisplatin and bleomycin chemotherapeutic drugs. Furthermore, only two cell lines displayed limited useful interactions of the CDU/5-FC either with interferon-β gene transfer or the proteasome inhibitor bortezomib respectively. These results would preclude a wide use of these combinations. However, the fact that all the tested cells were significantly sensitive to the CDU/5-FC system encourages further research as a gene therapy tool for local control of canine melanoma.

In the context of comparative oncology, melanoma cells derived from companion animal tumors are good models for optimizing and predicting their in vivo response to therapeutic strategies. Here, we report that human, canine, and feline melanoma cells driven to death by bleomycin, interferon-β gene, or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) treatment significantly increased their internal granularity. This fact correlated with the release of a heterogeneous collection of nano- and micro-sized granules as revealed by transmission electron microscopy. While killing lipofected cells, the expressed transgenes and their derived products were incorporated into these granules that were isolated by differential centrifugation. These particulate factors (PFs) were able to transfer, in a dose- and time-dependent manner, appreciable levels of therapeutic genes, related proteins, and drugs. Thus, when recipient cells of SG-carrying PFs were exposed to ganciclovir, this prodrug was efficiently activated, eliminating them. These PFs kept the functionality of their cargo, even after being subjected to adverse conditions, such as the presence of DNase, freezing, or heating. Since our in vitro system did not include any of the immune mechanisms that could provide additional antitumor activity, the chemo-gene treatments amplified by these delivery bags of therapeutic agents offer a great clinical potential.

After 6 years of follow-up treating 364 canine melanoma patients, we present here results about the proof-of-concept, safety, and efficacy of a new surgery adjuvant combined gene therapy. The adjuvant treatment (AT) group was divided in three arms as follows: (i) complete surgery plus vaccine (CS-V), (ii) complete surgery plus combined treatment (CS-CT), and (iii) partial surgery plus combined treatment (PS-CT). Besides the genetic vaccines composed by tumor extracts and lipoplexes carrying human interleukin-2 and granulocyte-macrophage colony-stimulating factor genes, the patients were subjected to combined treatment received in the post-surgical bed injections of lipid-complexed thymidine kinase suicide gene plus ganciclovir and canine interferon-β gene plus bleomycin. As compared with surgery-only treated controls (So), CS-CT and CS-V treatments significantly increased the fraction of local disease-free (from 20 to 89 and 74%) and distant metastases-free patients (M0: from 45 to 87 and 84%). Although less effective than CS arms, PS-CT arm demonstrated a significantly improved control of metastatic disease (M0: 80%) compared with So (M0: 44%). In addition, AT produced a significant 9.3- (CS-CT), 6.5- (CS-V), and 5.4-fold (PS-CT) increase of overall survival as compared with their respective So controls. In general terms, the AT changed a lethal disease into a chronic disease where 70% of CS-CT, 51% of CS-V, and 14% of PS-CT patients died of melanoma unrelated causes. These surgery adjuvant treatments delayed or prevented post-surgical recurrence and distant metastasis, and improved disease-free and overall survival while maintaining quality of life. These successful outcomes encourage assaying a similar scheme for human melanoma.

The incidence of canine mammary carcinoma varies with age, breed, and spay status, being among the main tumors appearing in intact female dogs. Thirty-six canine mammary carcinoma patients received injections of canine interferon-β (cIFN-β) and HSV-thymidine kinase/ganciclovir (HSV-tk/GCV) carrying lipoplexes, into the tumor bed, immediately after surgery. Next, they started periodic subcutaneous injections of lipoplexes carrying a human granulocyte-macrophage colony stimulating factor and interleukin-2 mixed with allogeneic mammary carcinoma extracts. This combined strategy was safe and well tolerated. In addition, only two out of 26 patients treated with complete surgery developed a local relapse, and 0 out of 29 stage II and III patients displayed distant metastases, suggesting both local and systemic antitumor activities. The most encouraging result was the long survival times: 22 > 1 year (where 13 > 2 and 4 > 3 years), while maintaining a good quality of life. The preliminary results in five patients presenting with local disease, an additional HSV-tk/GCV plus cIFN-β gene treatment induced local antitumor activity, evidenced by four objective responses (one complete, three partial) and one stable disease. This successful outcome supports further studies to validate this approach not only for canine veterinary patients, but also for translation to human patients.

We evaluated the cytotoxic effects of the combination of bortezomib (BTZ) and interferon-β (IFNβ) gene lipofection on cultured melanoma cells. Four canine mucosal (Ak, Br, Bk and Ol) and two human dermal (A375 and SB2) melanoma cell lines were assayed. BTZ sub-pharmacological concentrations (5 nM) enhanced the cytotoxic effects of IFNβ transgene expression on melanoma cells monolayers and spheroids. The combination was also more effective than the single treatments when assayed for clonogenic survival and cell migration. The combined treatment produced a significant raise of apoptosis evidenced by DNA fragmentation as compared to either BTZ or IFNβ gene lipofection single treatments. Furthermore, BTZ significantly increased the intracellular reactive oxygen species (ROS) generation induced by IFNβ gene transfer in melanoma cells, an effect that was reversed by the addition of the ROS inhibitor N-acetyl-L-cystein. The present work encourages further studies about the potential of the combination of interferon gene transfer with proteasome inhibitors as a new combined therapy for malignant melanoma, both in veterinary and/or human clinical settings.

This mini-review presents the results of veterinary clinical trials on immunogene therapy published from 2014 to 2016. A variety of tumors, among them melanoma (canine and equine), mastocytoma (canine), mammary adenocarcinoma (canine) and fibrosarcoma (feline) were treated by using diverse strategies. Non-viral vectors were usually employed to transfer genes of cytokines, suicide enzymes and/or tumor associated antigens. In general terms, minor or no adverse collateral effects were related to these procedures, and treated patients frequently improved their conditions (better quality of life, delayed or suppressed recurrence or metastatic spread, increased survival). Some of these new methodologies have a promising future if applied as adjuvant treatments of standard approaches. The auspicious results, derived from immunogene therapy studies carried out in companion animals, warrant their imperative usage in veterinary clinical oncology. Besides, they provide a strong preclinical basis (safety assays and proofs of concept) for analogous human clinical trials.