腸胃道間質腫瘤之致病機轉: 從早期發展至高惡性度轉變

Pathogenesis Of Gastrointestinal Stromal Tumors: From Early Development To High-Grade Transformation

梁哲維(Cher-wei Liang)

指導教授 ： 張逸良

國立臺灣大學/醫學院/病理學研究所/博士(2022年)

https://doi.org/10.6342/NTU202200766

摘要

腸胃道間質腫瘤（gastrointestinal stromal tumor, GIST）是腸胃道最常見的惡性非上皮性軟組織間質腫瘤，年發生率約介於每百萬人口6.5~14.5之間。但小於一公分之微小腸胃道間質腫瘤(microGIST)在一般成年人口的盛行率卻可達20~30%。這些須微觀才能發現之微小腫瘤，似乎是臨床可見腫瘤(大於一公分)之前驅病灶，故其可作為研究這類腫瘤之早期發生、演進及退化的良好模型。大部分的microGISTs，無法進一步生長至臨床可見的腫瘤而退化了。此研究利用回溯性案件搜尋找出了43例的microGISTs，證明了這些微小腫瘤無論在形態上、關鍵基因的突變上、基因的表現型、及蛋白質表現的模式上，均是與臨床可見的案例處在同一個光譜上。他們雖有著一樣促發腫瘤生成的KIT及PDGFRA特定基因的突變，但這也顯示了這些突變本身，並非腫瘤演進侵襲的主因。相同的，在臨床腫瘤演進中所發現的染色體變化，例如14q缺失、22q缺失、15q缺失、及1p缺失，一樣可以在microGISTs中發現，代表這些變化，雖然為腫瘤演進所必須，但單獨這些變化，並不足以使得GIST惡性轉變。經過全基因體複製的技術，也發現在臨床腫瘤內關鍵表現的生物標記，其基因諸如KIT、ANO1、PRKCQ、ETV1、SPRY4及PROM1，在microGISTs內也一樣被活躍的轉譯中。藉由突變功能性測試、免疫螢光染色、及mRNA原位雜交法，發現在microGISTs中發生了比例較高之特定KIT突變型，其產生之異常KIT接受體(KIT receptor)對於KIT配位基(KIT ligand)的敏感度較高，而這些KIT配位基主要是由腸胃道之平滑肌壁細胞所分泌。故當腫瘤成長之大小超過周邊平滑肌可供應配位基的範圍時，其下游的AKT/S6路徑將會被抑制不再活化，進一步導致了腫瘤的退化。
接下來，計畫繼續探討若腫瘤獲得了繼續演進生長的能力，那麼其高惡性度轉變的關鍵步驟為何。研究中發現有少量具有雙相特徵的GISTs (biphasic GISTs)，即在同一個腫瘤中具有相鄰的低惡性度區域及高惡性度區域，代表腫瘤剛好在高度惡性轉變期間被手術所擷取。計畫執行期間總共回溯性搜尋了263例GISTs，並從其間找出了19例的雙相腫瘤。研究亦另外涵蓋了82例橫跨各惡性度的一般GISTs做為比對之用。在進行了基因突變、螢光原位雜交(fluorescence in situ hybridization, FISH)、NanoString基因數平台、端粒酶(telomere)、及基因表現量之分析後，進一步使用資料庫分析(in silico)、細胞株、及免疫化學染色來做進一步的確認。在基因表現量的分析上，發現SFRP1之調降為GIST高惡性度轉變的關鍵步驟(p = 0.013)。此現象亦伴隨著EZH2之調升。分析已被釋出的腫瘤資料庫內的案例，發現在不同的系列中，SFRP1之調降是一個可以被普遍驗證的事項。利用免疫組織化學染色做再驗證，發現SFRP1蛋白的表現量，在高惡性度的GISTs中(WHO風險分群3a級或以上)，有明顯降低的現象(p < 0.001)。而SFRP1蛋白表現的減弱或喪失，正比於GIST演進之惡性度。利用NanoString基因數平台及FISH進行分析，發現在雙相腫瘤中，染色體9及9p的缺失，是在腫瘤高惡性度轉變中，唯一有被發現反覆出現的事件，而且此缺失現象，亦與SFRP1之調降相關。另在螢光檢查中，可在雙相腫瘤中之低惡性度區發現有染色體9及9p缺失的次族群(subclones)。TP53基因突變、RB1基因缺失、KIT/PDGFRA基因突變、及端粒酶之替代性延展(alternative lengthening)，則沒有發現與腫瘤高惡性度轉變有重要的相關。總結上，SFRP1的調降及染色體9/9p之缺失，為GIST高惡性度轉變的關鍵步驟。

關鍵字

腸胃道間質腫瘤(GIST) ； 微小腸胃道間質腫瘤(microGISTs) ； KIT ； PDGFRA ； 全基因體複製 ； SFRP1 ； 染色體9/9p

並列摘要

Gastrointestinal stromal tumor (GIST) is the most common malignant mesenchymal tumor in the gastrointestinal tract with an annual incidence rate ranging from 6.5 to 14.5 per million, and benign microGISTs (less than 1 cm in diameter) are found in 20-30% of the general adult population. These microGISTs seem to be precursor lesions of clinical GISTs and are therefore compelling models in which to characterize early transforming, progressing, and involuting mechanisms. Most of the microGISTs involute and fail to progress. Herein, we collected 43 microGISTs by retrospective archive screening and demonstrated that they were on a biological spectrum with clinical GISTs, as judged by morphologic considerations, key oncogenic mutations, gene expression profiles, and protein expression patterns. One subset KIT and PDGFRA mutations, as initiating events shared by clinical and microGISTs, per se are not, in and of themselves, biological mechanisms of aggressive behavior, and cytogenetic aberrations, such as 14q loss, 22q loss, 15q loss and 1p loss revealed by array comparative genome hybridization in microGISTs, are necessary, but not sufficient, in genetic progression towards GIST malignancy. By whole-genome amplification, we detected crucial GIST biomarkers (e.g., KIT, ANO1, PRKCQ, ETV1, SPRY4 and PROM1) being actively transcribed in microGISTs, representing early events. By construct mutagenesis functional test, immunofluorescence and mRNA in situ hybridization, we also demonstrated that a subset of microGISTs carrying certain types of mutations that could sensitize KIT receptor to KIT-ligand stimulation from smooth muscle cells, which supported early microGIST development. In involution, microGIST cells outstrip available KIT-ligand with resultant inhibition of AKT/S6 pathways.
The mechanism of high-grade transformation in GIST then remains to be clarified. We aim to discover the key progression events by studying biphasic GISTs. The study group included 101 GISTs. Nineteen of these had been screened from 263 GISTs to represent the early stage of GIST high-grade transformation, characterized by juxtaposed low-grade and high-grade regions in the same tumor (so-called biphasic GISTs). Mutational analyses, fluorescence in situ hybridization (FISH), NanoString analyses, telomere analysis and gene expression profiling were done, followed by in silico analyses, cell line study, and immunohistochemical validation. Utilizing gene expression analysis, downregulation of SFRP1 was revealed to be the main event in GIST high-grade transformation (p = 0.013), accompanied by upregulation of EZH2. In silico analyses revealed that downregulation of SFRP1 was a common feature in GIST progression across several different series. Immunohistochemically, the expression of SFRP1 was validated to be significantly lower in high-grade GISTs (WHO risk group 3a or higher) than in lower-grade GISTs (p < 0.001), and attenuation/loss of SFRP1 was associated with GIST tumor progression (p < 0.001). By NanoString and FISH analyses, chromosomal 9/9p loss was the only recurrent large-scale chromosome aberration in biphasic GISTs, with a correlation with SFRP1 downregulation. Subclones containing chromosome 9/9p loss could be appreciated in the low-grade parts of biphasic GISTs. TP53 mutation, RB1 loss, KIT/PDGFRA mutation and alternative lengthening of telomeres did not play a significant role in GIST high-grade transformation. In conclusion, high-grade transformation of GISTs features SFRP1 downregulation and chromosome 9/9p loss.

並列關鍵字

gastrointestinal stromal tumor (GIST) ； microscopic GISTs (microGISTs) ； KIT ； PDGFRA ； whole-genome amplification ； SFRP1 ； chromosome 9/9p

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