不同治療方式於犬口腔惡性黑色素瘤之應用

Abstract

黑色素瘤是犬口腔中最常見的惡性腫瘤，有極強的局部侵犯性和遠端轉移能力，使得臨床治療非常具有挑戰性。目前針對犬口腔惡性黑色素瘤最有效的治療方式仍是局部控制，包含大範圍手術和放射線治療。而傳統治療方式包含化學治療及標靶治療的臨床效果則較有限。免疫治療作為近年來的癌症治療第四支柱，在犬腫瘤中已經有所探索。然而目前在台灣仍存在一些困境，例如缺乏專為動物使用的放射治療、商品化的犬免疫治療如Oncept®和Gilvetmab®尚未進口，有時對於犬口腔黑色素瘤甚至局部大範圍切除都無法實現。因此，本論文整體目標為研究口腔惡性黑色素瘤患犬的可能的治療方式。 本研究包含三個部分，從傳統化療效果分析到新型免疫治療評估。首先第一個部分，為回溯性分析一組未接受大範圍手術切除和放射治療的黑色素瘤患犬，使用化學治療的效果，因目前對於此缺乏良好局部控制的群體，化療仍是僅剩的選項。第二個部分為一項自體免疫細胞回輸的初步研究。同樣收集口腔惡性黑色素瘤患犬，研究此治療的安全性，因此沒有腫瘤相關的排除標準。第三個部分為一項以樹狀細胞為基礎的臨床試驗，使用異體樹突狀細胞（DC）和自體腫瘤細胞製作融合疫苗，將口腔惡性黑色素瘤患犬分為疫苗單獨組（DC組）、與放射治療結合組（DCRT組），同時從第一項研究中提取一組接受carboplatin治療的犬隻作為對照組，探討融合疫苗單獨使用及與放療結合的安全性和初步臨床效果。 第一項研究共納入13隻口腔惡性黑色素瘤患犬，化療方案由臨床醫師決定，包含carboplatin（n=8）和鐘擺化療（n=5）。所有犬隻都未接受大範圍手術或放療，但有些狗接受了腫瘤減積手術。研究統計得到的中位無疾病進展時間（PFI）為42天（95% CI, 20-69），僅接受化療的犬隻的中位存活時間（OST）為181天（n = 11；95% CI，NA-350）。使用carboplatin的中位劑量為250 mg/m²。處於較早的臨床分期和化療期間腫瘤至少維持穩定，是顯著較好的預後因子。第二項研究共收集了10隻患犬接受自體免疫細胞回輸。回輸所用細胞為從患犬自體身上的周邊血液提取所得，通過細胞因子IL-15、IL-2和IL-21的組合刺激在體外擴增為non-B non-T細胞，再通過緩慢靜脈給於的方式回輸到患犬體內。臨床上，有三隻犬接受治療後未發生任何副作用；三隻犬出現了輕微的食慾改變；一隻犬出現肝指數輕微升高；而另外三隻犬出現了不同程度的疑似過敏反應。此研究統計所得的中位無疾病進展時間為49天。在治療期間發生腫瘤進展是顯著較差的預後因子。第三項研究同樣為前驅性免疫治療臨床試驗，以樹狀細胞融合疫苗為基礎。五隻患犬被納入DCRT組，十一隻納入DC組，控制組有八隻。DC疫苗每兩週施打一次，共四次。放射治療每週進行，共五次。結果顯示DC疫苗單獨使用或結合放療均耐受良好，僅有輕微的副作用發生，包含黏膜發炎、消化道不適和局部注射反應。DCRT、DC和控制組的中位無疾病進展時間分別為214天（95% CI，NA）、100天（95% CI，27-237）和42天（95% CI，NA-170），三組未有顯著差異，1年生存率分別為20%、54.5%和12.5%。DCRT組的犬隻和DC組相比，在整個治療過程中皆顯示出顯著更高的TGF-β濃度，意味著可能存在更高程度的免疫抑制。 這三項研究描述了在台大接受治療的口腔惡性黑色素瘤患犬從過去到現在的治療選項變化和發展。總體而言，化療能提供了中位6個月的生存時間，當局部控制無法實現時，化療仍是可考慮的選項。同時，兩項初步免疫治療研究，自體細胞回輸和樹狀細胞融合疫苗，在犬類癌症治療中具有創新性。自體細胞回輸的臨床效果相對有限且可能存在副作用，樹狀細胞融合疫苗相對安全。這兩項研究的初步結果支持未來針對這兩項治療，不論是單獨使用還是結合其他的治療方式，進行進一步的治療療程優化以及設計更嚴格的臨床試驗。同時，對免疫治療期間的免疫相關因子進行全面的研究也值得進行，以更好地理解免疫治療過程中的故事。

Melanoma is the most common malignancy in the canine oral cavity. Its great propensity for local invasion and distant metastasis renders treatment challenging. Adequate local control with curative-intent surgery and radiation therapy provides the most effective tumor management, while conventional systemic treatments of chemotherapy and targeted therapy yield limited clinical benefit. Immunotherapy exhibits promising prospects in cancer treatment and has already been explored in canine species. However, some dilemmas exist in Taiwan, including the unavailability of exclusive animal-use radiation therapy and the commercialized canine immunotherapies Oncept® and Gilvetmab®, and sometimes wide-margin surgery cannot be achieved in the oral cavity. Therefore, this dissertation aims to explore possible treatment modalities in melanoma dogs in Taiwan. Three sub-projects were carried out, from conventional chemotherapeutic effect analysis to novel immunotherapies evaluation. Firstly, a group of dogs lacking wide-margin surgery and radiotherapy who received MTD or metronomic chemotherapy was retrospectively enrolled, because for a population without adequate local control, we do not have other options except for chemotherapy. Secondly, a pilot study of adoptive autologous immune cell transfer was conducted in dogs with oral malignant melanoma. There were no tumor-related exclusion requirements in this sub-project and the primary aim was to investigate treatment safety. Thirdly, another clinical trial of using allogeneic dendritic cell and autologous tumor cell fusion vaccine, alone (DC group) or in combination with radiation therapy (DCRT group), was executed. A group of dogs (control group) who received carboplatin was extracted from the first project as a control group. This sub-project aims to investigate the treatment safety of the fusion vaccine alone and in combination with radiotherapy, and the treatments’ efficacy. Thirteen client-owned dogs were enrolled in the first sub-project. Chemotherapy protocols were determined by the attending clinician, including carboplatin (n = 8) and metronomic chemotherapy (n = 5). None of them received wide-margin surgery or RT, but some had tumors marginally excised. The median PFI was 42 days (95% CI, 20-69), and the median OST of dogs having chemotherapy as their only systemic treatment was 181 days (n = 11; 95% CI, NA-350). The median dosage of carboplatin was 250 mg/m2. Earlier clinical stages and achieving at least stable disease during chemotherapy were significant positive prognostic factors. As for the second sub-project of the pilot adoptive cell transfer, ten dogs were enrolled. The infusion cells were generated autologously from the tumor-bearing dogs and were expanded ex vivo into non-B non-T cells through cytokine cocktail stimulations of IL-15, IL-2, and IL-21. The expanded infusion cells were administered slowly intravenously. Clinically, three dogs did not report any adverse events; three had a mildly altered appetite; one had a mildly increased liver index; while the other three developed suspected anaphylaxis at different levels. The median PFI was 49 days. Dogs with progressive disease during treatment had a shorter survival. The third sub-project was also a pilot clinical trial evaluating dendritic cell-based treatment. Five dogs were included in the DCRT group, eleven in the DC group, and eight in the control group. DC vaccination was given once every two weeks for four doses. Radiotherapy was performed weekly for five fractions. Both DC and DCRT were well-tolerated, with only mild adverse events reported, including mucositis, gastrointestinal discomfort and injection site reactions. The median PFIs in the DCRT, DC and control groups were 214 (95% CI, NA), 100 (95% CI, 27-237), and 42 days (95% CI, NA-170), respectively, which were not significantly different, and the 1-year survival rates were 20%, 54.5%, and 12.5%. Dogs in the DCRT group exhibited significantly higher TGF-β signals throughout the treatment course, indicating a possible higher degree of immunosuppression. The results of the three sub-projects described the picture of systemic treatment options and developments in our department for canine oral melanomas. Generally, chemotherapy offered a median survival of 6 months and could still be considered when adequate local control is infeasible. Meanwhile, the two pilot immunotherapeutic studies, the adoptive cell transfer and the DC-based fusion vaccine, are innovative in canine cancer treatments. The adoptive cell transfer demonstrated possible adverse events and limited clinical benefit, while the DC fusion vaccine was safe and potentially effective. The preliminary findings of these two pilot studies support further protocol optimization and exploration, and necessitate more rigorously designed clinical trials of either using these treatments alone or in combination with other therapies. Investigation of a comprehensive immune parameter complex during immunotherapy is also worthwhile, to have a better understanding of immune modulations after immunotherapy.