運用動態顯影磁振造影於臨床與基礎研究:鑑別脊椎病理性骨折、實施腫瘤分子標靶影像並早期評估抗血管新生藥物治療之反應

Dynamic contrast enhanced MRI in clinical and basic researches: differentiating vertebral pathologic fracture, tumor targeting molecular imaging, and early assessment of anti-angiogenic treatment response

陳慰宗(Wei-Tsung Chen)

指導教授 ： 施庭芳

共同指導教授 ： 楊泮池(Pang-Chyr Yang)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.00660

摘要

本論文分為臨床研究與基礎研究兩部分，以動態顯影磁振造影為切入點。在臨床研究方面，我們利用動態顯影磁振造影具有偵病灶血管新生並顯示組織內血液灌流現象的能力，找出良性與病理性脊椎壓迫性骨折病灶中骨髓血液灌流模式的差別，此發現對於某些困難診斷的脊椎壓迫性骨折提供了一個有效的解決方法。這在患者沒有惡性腫瘤的病史情形下尤其重要，因我們若是將病理性脊椎壓迫性骨折誤診為良性骨折，將延誤治療時機，增加日後治療的困難度。
在此研究過程中，我們同時發現並證實了脊椎骨髓血液灌流生理現象與老化的關係:年齡越大、其骨髓血液灌流的程度就越低，且脊椎骨髓血液灌流在女性隨年齡下降的更快。因著上述發現，我們又推想骨髓血液灌流的程度多寡會不會與動脈硬化的程度有關? 在一個43對性別年齡配對研究中,我們將性別和年齡這兩個重要的干擾因子控制住，證實了內頸靜脈內膜厚度越大，脊椎骨髓血液灌流就越少。另外我們也同時提出了脊椎骨髓內脂肪水分比例與脊椎骨髓血液灌流具有負相關性的證據。這一系列發現使我們懷疑骨髓血液灌流與老年人的大敵”骨質疏鬆”是否有關聯。事實上，在指導教授施教授後續的研究中，也證實了女性骨髓血液灌流越低，其骨質密度也越低。故將來在討論骨質疏鬆的機轉時，組織缺血也可能是一重要影響因子。
在基礎研究方面，我們利用動態顯影磁振造影配合標靶顯影劑來偵測組織或病灶內特殊分子標記的表現。在實驗中我們先成功將多個葉酸以及磁振造影T1顯影劑Gd-DTPA接合在以聚二乙醇為主幹的分子載體上，使其成為可在生物體內尋找葉酸接受體的磁振標靶顯影劑，並以動態顯影磁振造影觀察到此顯影劑如何流進與流出在表現葉酸接受體腫瘤內的情形，並和不表現葉酸接受體之腫瘤做對照。這個研究證明了使用大分子載體接合傳統磁振造影顯影劑與一些對腫瘤標記有高親和力的分子探針來從事磁振造影標靶影像的可行性。這種方法不僅讓本來無特異性的磁振造影顯影劑在體內增加了尋找腫瘤標記的能力，同時因一個分子載體上接合了多個Gd-DTPA，而同時達到將磁振造影訊號放大的目的，因而可提高磁振造影的敏感度。一般Gd-DTPA的Ｔ1鬆弛度為2.9(mM S)-1，此葉酸接受體標靶顯影劑的Ｔ1鬆弛度為　53.2(mM S)-1，此標靶顯影劑之顯影能力比傳統磁振造影顯影劑多了18.3倍。
在論文第三部份：以動態顯影整合素integrin αvβ3標靶磁振造影影像偵測腫瘤血管新生並早期評估抗血管新生藥物治療後之反應。我們也再次證實了這個概念，並將其推廣到早期評估抗血管新生藥物治療療效。在本論文的第一部分我們證實了動態顯影磁振造影具有偵測病灶血管新生與分析其內血液灌流的能力，但已達到目前傳統磁振造影能力的極限。在第二、三部分我們配合磁振標靶顯影劑擴展了目前磁振造影的能力，不僅證明了接合上環狀RGD胜肽的分子載體在細胞層級具有吸附表現整合素integrin αvβ3的U87細胞的能力；我們更在活體影像中，以動態顯影磁振造影以及所產生的參數”三十分鐘顯影劑流失比率”證實此分子探針會因著吸附在活化內皮細胞上的整合素integrin αvβ3而有較長的時間停留在有腫瘤血管新生的部位，而被磁振造影偵測出來。實驗中所有腫瘤若使用此標靶顯影劑，其”三十分鐘顯影劑流失比率”皆為負值(但若使用對照組接合環狀RAD胜肽的顯影劑此現象即消失)。 但是在腫瘤開始接受抗血管新生標靶藥物治療的實驗後，（包括使用anti-integrinαvβ3 monoclonal Ab及anti-VEGF monoclonal Ab），即使使用環狀RGD胜肽的顯影劑，腫瘤的”三十分鐘顯影劑流失比率”也變為正值，代表此標靶顯影劑在三十分鐘內就會從腫瘤流失；而我們在十多天後才觀察到腫瘤體積開始縮小。本實驗結果證實了標靶磁振造影加上動態顯影磁振造影所提供的”三十分鐘顯影劑流失比率”，不僅可以用來確認病灶內有分子標記的表現，更可以在腫瘤大小發生改變以前得知腫瘤已發生治療反應，這是目前影像醫學尚無法達到的。
總而言之，本論文一方面以動態顯影磁振造影在臨床分析病灶血管新生血液灌流模式成功鑑別了病理性脊椎壓迫骨折與良性壓迫性骨折；一方面在基礎研究以動態顯影磁振造影配合標靶顯影劑成功實施了葉酸接受體標靶影像和整合素αvβ3標靶影像並將其應用於早期評估抗血管新生藥物治療之反應，本研究之發現對未來磁振造影分子影像與臨床癌症治療規劃決策將產生很大衝擊。

關鍵字

動態顯影磁振造影 ； 血液灌流 ； 壓迫性骨折 ； 分子影像 ； 葉酸接受體 ； 血管新生 ； 整合素αvβ3 ； 抗血管新生治療

並列摘要

Clinical (part 1) and basic researches (part 2, 3) were included in this thesis. By using dynamic contrast enhanced (DCE) MRI, we first demonstrated its ability for revealing hemodynamic changes in vertebral compression fractures, and successfully differentiated pathologic form benign vertebral compression fracture by analyzing the blood perfusion patterns and parameters. We established an effective and easy way for the diagnosis of pathologic vertebral compression fracture in cases with equivocal conventional MR findings. This is valuable when the primary malignancy is not known by the patient or physicians. If the pathologic fractures are misdiagnosed, the proper treatment timing will be delayed.
We also found an interesting issue of the relationship between the vertebral blood perfusion status and aging process. We noticed that the older our patients, the lower the bone marrow blood perfusion degree, revealed by DCE MRI. To validate this finding, we analyzed DCE MRI data from 60 subjects with varied age in both genders, and found a significant negative correlation between vertebral marrow blood perfusion and aging, this phenomenon was especially noticeable in female subjects.
According to above findings, we hypothesized that there might be a correlation between atherosclerosis and marrow blood perfusion. A sex- and age- matched study was proposed. We selected patients whose internal carotid artery intemal thickness was larger than 1mm from a database of 2245 patients who received carotid artery ultrasonography in our hospital, once a thick IMT subject was included in this study, another subject was selected with normal IMT from the same database under sex- and age matched policy. Totally 43 sex- an age- matched pairs were included in this study. Out results showed a significant negative correlation between internal carotid intemal thickness and marrow blood perfusion.
We also noticed that the fatty marrow deposition progressed with aging process, and hypothesized that the lipid ratio within bone marrow might have a correlation with marrow blood perfusion. MR spectroscopy was used to analyze MRS data from 50 female subjects to reveal the relationship between the marrow lipid water ratio and marrow blood perfusion, and a significant negative correlation between marrow blood perfusion and marrow lipid water ratio was proven.
In the clinical research portion, we first developed an easy and reliable method to differentiate pathologic vertebral compression fracture from osteoporotic fracture, by analyzing the blood perfusion patterns. Furthermore, we evaluated the vertebral marrow blood perfusion phenomenon with aging, atherosclerosis and marrow lipid water ratio, such relationships were not explored in the literature. Our results lead us to hypothesize that there is a link between marrow blood perfusion and osteoporosis, which is an important issue in aging medicine. In later studies, professor Shih demonstrated that there is a positive correlation between bone mineral density and marrow blood perfusion. This finding implied that ‘ischemia’ might be an important factor related to osteoporosis.
In the basic research portion, the aim of our study was to use DCE MRI combine with receptor-targeted MRI contrast medium to detect molecular signatures in tumors. We first successfully synthesized a folate conjugated, Gd-DTPA loaded polyethylene glycol (PEG) dendrimer, as a folate receptor (FR) targeted contrast medium. We observed hemodynamic changes of FR positive and FR negative tumors in living mouse DCE MR imaging. Our results showed that the conjugation of molecular ligand to a carrier loaded with Gd-DTPA is feasible for receptor targeted MR imaging.
This methodology not only turned a non-specific MR contrast medium into a receptor specific MR contrast agent, but also had the advantage of MR signal magnification, but adding multiple Gd-DTPA onto a dendrimer, hence, the sensitivity of this receptor contrast medium will increase. The T1 relaxicity of Gd-DTPA was 2.9(mM S)-1，however, the T1 relaxicity of the FR targeted contrast medium was 53.2(mM S)-1, which was 18.3 fold higher.
In the third part of the thesis, we demonstrated that the cRGD conjugated contrast medium can attached to the cell surface of the integrin αvβ3 expressing U87 MG in cellular attachment experiments; we also showed the targeting ability of this integrin αvβ3 targeting contrast medium in living animals, by using DCE MRI. The 30-min contrast washout percentage derived form DCE MRI showed that the integrin αvβ3 targeting contrast medium stayed longer in sites of tumor angiogenesis. If we used cRAD conjugated contrast medium as a control drug, in stead of RGD domain, the persistent contrast enhancement phenomenon was disappeared. However, in the anti-integrin αvβ3 monoclonal antibody or anti-VEGF monoclonal antibody studies, the persistent contrast enhancement phenomenon was disappeared, and a rapid contrast washout pattern was observed rapidly following the anti-angiogenetic therapy, even before the tumor volume decreased, which was observed 10 days after the start of anti-angiogenetic therapy. We confirmed our hypothesis, i.e., receptor targeted MR imaging was achievable and can be used in early assessment of target therapy, even in advance to tumor size decrease. Such ability is beyond any present medical imaging modalities.
In conclusion, we have demonstrated the clinical application of DCE MRI for differentiating pathologic vertebral compression fracture. The ability of DCE MRI is reinforced by using receptor targeting MR contrast medium. We have not only demonstrated the ability of folate receptor and integrin αvβ3 targeting conjugates in vitro and in vivo, but also showed its superior ability in the early assessment of anti-angiogenic therapy. Our findings will bring important impacts for future MR molecular imaging and clinical cancer treatment strategies.

並列關鍵字

dynamic contrast enhanced MRI ； blood perfusion ； compression fracture ； molecular imaging ； folate receptor ； angiogenesis ； integrin αvβ3 ； anti-angiogenic therapy

參考文獻

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