犬癌症之免疫治療與影像診斷

Immunotherapy and imaging diagnosis of canine cancers

莊添富(Tien-Fu Chuang)

指導教授 ： 廖泰慶 ； 朱瑞民

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.01561

摘要

癌症是引起動物死亡的主要原因之一。在犬癌症中，乳腺腫瘤是母犬最常見的腫瘤，占所有腫瘤發生率的50％，在組織學診斷中，發現為惡性乳腺腫瘤的機率為50％。口腔惡性腫瘤中，黑色素瘤是最常見的腫瘤，是一個極端侵略性的腫瘤，發生率為20%-50%。手術切除是乳腺腫瘤和黑色素瘤最主要的治療方式，但是預後不佳，容易復發或轉移。其他輔助治療，例如化療、放射線治療或賀爾蒙治療都無法有效增進手術的效果，因此，在本篇論文中，我們針對惡性乳腺腫瘤和黑色素瘤發展新的治療策略，期望改善腫瘤病犬的預後與生活品質。
首先，在惡性乳腺腫瘤治療研究上，依據最近的標把治療和免疫治療，我們設計了異種Her2 DNA疫苗（xenogeneic Her2 DNA vaccine）輔助手術治療犬惡性乳腺腫瘤，總計有29隻患有惡性乳腺病犬納入研究中，分為兩組：一組為控制組（只有進行手術移除），一組為Her2組（手術與疫苗合併治療）。此DNA疫苗給予的方式為2次皮內注射加上1次肌肉電衝， 在臨床上，異種DNA疫苗可以克服Her2自我抗原在動物體內的免疫耐受性，刺激特異性ELIspot IFN-γ反應。結果顯示，DNA疫苗輔助手術可以抑制腫瘤生長和遠端轉移，延遲手術後原位復發。在Her2組，此疫苗明顯延長病犬的存活時間，平均為678天（212-779天），控制組為249天(142-573天)，除此之外，此DNA疫苗對於病犬無任何副作用，在犬惡性乳腺腫瘤，此異體DNA疫苗合併手術切除治療惡性乳腺腫瘤，為一個具有高度潛力的治療方式。
其次，因為在惡性黑色素瘤治療研究上，還未發現以骨髓來源樹狀細胞/腫瘤融合疫苗治療犬口腔黑色素瘤的文獻，於是我們進一步評估樹狀細胞/黑色素瘤融合疫苗在犬口腔黑色素瘤的治療效果。我們也比較單獨治療或者手術與疫苗合併治療在臨床上的效果。一共有31隻犬被納入此研究中，此31隻犬全為口腔黑色素瘤第三期，被分成三組：一組為控制組（只有進行手術移除，SR），一組為單獨進行疫苗治療（VO），一組為手術與疫苗合併治療組（CT）。在合併治療組（CT），疫苗可以明顯延長犬存活時間為259天（90-350天)，在疫苗單獨治療組（VO）為124天（43-231天) 和單獨手術組（SR）為82天（50-140天)。更進一步，合併治療可以顯著降低轉移率，從單獨手術組的56％降低至合併治療組的30％，且延遲原位復發。更可以增強腫瘤特異性T細胞毒殺能力，在合併治療組為30.4% ± 14.6%，在單獨治療組為20.1% ± 9.5%，治療前則為5.7% ± 2.7%。合併治療組中，犬周圍血液中的Treg細胞明顯的下降。此融合細胞疫苗對病畜是安全的。這些研究結果指出樹狀細胞/腫瘤融合疫苗與手術合併使用對於口腔黑色素瘤是有效的治療策略，可以改善病畜的生活品質。此合併治療策略可以考慮用於臨床口腔黑色素瘤的治療或其他哺乳動物的癌症治療。
最後，我們發展新的技術去偵測免疫治療的反應，使用影像學診斷與傳統的解剖學變化相比，可以更快速偵測到免疫反應，也比組織學的方式更無侵入性。在過去的癌症免疫學治療中，PET/CT已經使用在小鼠模式中去評估抗腫瘤反應，但是在較大型動物模式（犬模式）中很少有研究去監控腫瘤免疫治療的效果。犬傳染性花柳性腫瘤是唯一可以在犬身上人工接種的腫瘤，是最佳的犬腫瘤模式，我們之前已經發表過使用腫瘤內電衝IL-12基因，會導致CTVT腫瘤消退且明顯吸引大量腫瘤浸潤淋巴球，於是在此研究中我們更進一步使用FDG-PET去偵測此免疫治療的反應，我們可以發現CTVT腫瘤的代謝是可以被FDG-PET偵測且腫瘤變化可以在早期就被發現。我們測試了不同的時間，發現在FDG注射後的60分鐘，FDG的吸收率達到穩定的狀態，此外，IL-12免疫治療將導致每單位體積較高的FDG吸收率，此趨勢可以在CTVT接種後第19天被偵測到，而給予病犬IL-12與FDG並無任何毒性產生，FDG-PET/CT可以早期偵測免疫治療對腫瘤造成的真實變化，因此我們可以推測此免疫治療是否為成功的治療，隨時操控治療策略，將可以提高治療的成功率。
犬癌症的研究可以在小鼠臨床前研究與人類臨床試驗間扮演一個重要橋樑，促進獸醫與人類癌症共同研究，這樣模式的研究將導致犬癌症與人類癌症的治療成果蓬勃發展。

關鍵字

犬 ； 乳腺腫瘤 ； 黑色素瘤 ； 免疫治療 ； 融合疫苗 ； 正子照影

並列摘要

Cancer is the most common disease-associated cause of death or euthanasia in older canines. Among canine cancers, mammary gland tumor (MGT) is the most common tumor in female canines accounting for near 50% of all tumors and around 50% of those MGT is malignant. The oral melanoma accounted for 20% to 50% of oral malignancies and has been routinely considered an extremely aggressive tumor. So far, surgical excision is the most widely accepted treatment for MGT and oral melanoma, however, it offers poorly on long term prognostic or preventing metastasis in malignant cases. Other adjunctive therapies, such as chemotherapy, radiation or hormone therapy, cannot effectively promote the therapeutic effect of surgical removal. In this thesis, we looked for new approaches to therapy malignant MGT and oral melanoma and to achieve a good quality of life in canine patients.
First, according to recent achievement in targeted therapy and immunotherapy, we applied a recombinant rat Her2 xenogeneic DNA vaccine, pING-rHer2, to treat canine malignant MGTs following surgical removal of the tumor masses. Twenty-nine canines with MGT were enrolled in this program and were divided into two groups, control (surgical removal only) and Her2 (combined treatment, surgery plus vaccination). The DNA vaccine was delivered by two transdermal injections followed by an intramuscular electroporation. The antigen-specific ELIspot IFN-γ response elicited by pIGN-rHer2 DNA vaccinations was a clear demonstration that this vaccine was able to overcome tolerance to the Her2 self antigen under clinically relevant conditions. Furthermore, the results demonstrated that surgery combined with pING-rHer2 DNA vaccination significantly restrained the tumor growth and distant mestastasis, and substantially postponed post-surgical recurrence in canine spontaneous malignant MGT. The vaccine significantly prolonged the patients overall survival time in the Her2 group. In addition, there was no detectable side effect. In conclusion, this xenogeneic DNA vaccine should be further considered as a potential immunotherapy of canine malignant MGT.
Second, because the use of an allogeneic bone marrow-derived dendritic cell (BMDC)/tumor cell fusion vaccine to treat canine oral malignant melanoma has not been documented and the efficacy of the BMDC/syngeneic melanoma cell fusion vaccine was evaluated in canine oral melanoma patients. We also compared the clinical benefits for patients between single treatments and a combination protocol of surgery/fusion vaccine. Thirty-one canines with stage III oral melanoma were enrolled in this program and were divided into three groups, SR (surgical removal only), VO (vaccination only) and CT (combined treatment, surgery plus vaccination). The vaccine significantly improved the overall survival time in the CT group (259 days; 90–350 days) in comparison with the VO (124 days; 43–231days) and SR groups (82 days; 50–140 days). Moreover, the CT protocol dramatically decreased the metastatic rate and delayed in situ recurrence; it also stimulated potent tumor cell-specific cytotoxic T lymphocytes (CTL). In addition, the population of Treg cells in peripheral blood decreased significantly in the CT canines in comparison with the other groups. Besides, there was no clinically-detectable toxicity. These results indicate that surgical removal combined with the allogeneic BMDC/syngeneic tumor cell fusion vaccine was the effective treatment protocol for late-stage canine oral melanoma and resulted in an improvement in quality of life. It is suggested that this combined protocol can be considered one of the treatments of choice for canine oral melanoma and has the potential to be used in other mammals.
Furthermore, we have developed new image technique to indicate immunotherapy response sooner than anatomic changes depicted by traditional modalities, and less invasively than histological sampling. Regarding immunotherapy of cancer, PET/CT has been used in mice models to evaluate antitumor response. There are few researches in monitoring the efficacy of cancer immunotherapy in larger animal models such as canines using PET/CT. Canine transmissible venereal tumor (CTVT) can be experimentally transplanted to allogeneic canines. CTVT is an ideal model for cancer study. Previously, we have publisded that intratumoral electroporation of pIL-12 resulted in successful tumor regression and attracted significantly more lymphocytes infiltrating to the tumors in the canine CTVT model. CTVT metabolism can be measured by serial FDG-PET scansand the change of tumor in the early phase can be detected. At 60 min after FDG delivery, there was a stable trend of FDG uptake ratio in CTVT. The pIL-12 immunotherapy resulted inhigher FDG uptake ratios per volume of CTVT, which can be detected as early as 19 days after CTVT inoculation. Delivery of pIL-12 and FDG did not result in any detectable toxicity in the canines. The FDG PET/CT early detect the tumor growth pattern. The ability of FDG PET/CT to measure tumor growth directly after the commencement of treatment may provide an early indicator of the success of therapy. Immediately, the strategies of treatment will be managed. The FDG PET/CT will improve the diagnosis of the efficacy and successful rate of future immunotherapy clinical trials in cancer patients. Therefore, these protocols have the potential to be used in other canine cancers.
Canine cancers may serve as an important bridge between preclinical studies in mouse model systems and clinical trials in humans with cancer and support the synergy of collaborations between veterinary and human cancer centers. Such collaboration will result in the development of therapy in canines cancers and has shown promising results in humans.

並列關鍵字

Canine ； Mammary gland tumor ； Oral melanoma ； Immunotherapy ； Her2 DNA vaccine ； Fusion vaccine ； FDG PET/CT

參考文獻

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