基因融合 ( gene fusion ) 是癌症中一類重要的基因病變，在腫瘤發生的初期即發揮重要作用，約佔人類罹癌發病的 20%，相對於其他突變類型的研究，卻沒有足夠豐富的資料，加上融合基因發生在各個癌症/病況之間的差異很大，外顯子分析可就基因編碼區域做掃描，快速找到多個 fusion 位點，對於未知新穎、待研究的罕見癌症，確可助於有效歸納、累積癌症 gene fusion 資料。
2022 年路易斯維爾大學布朗癌症中心 ( University of Louisville / James Graham Brown Cancer Center ) 在 Nature Communications 期刊上發表了星形芽細胞瘤 ( Astroblastomas, ABs ) 的基因研究，ABs 為一種罕見、起源不明的上皮性腦腫瘤，通常發生在年輕人和兒童，女性居多。目前知道 ABs 至少有兩種基因型，分別是 BRAF V600E 突變型與 MN1 基因重組型，其中 MN1 基因重組型的腫瘤被報告過存在 MN1-BEND2 融合基因。
布朗癌症中心收集了 24 例類 AB 腦瘤 ( AB-like tumors, 6例 BRAF V600E, 6例 MN1-重組, 12例不具有 BRAF V600E 或 MN1-重組 )，及 7 例的天幕上室管膜瘤 ( supratentorial ependymoma, STE ) 或松果體乳頭狀瘤 ( papillary tumor of the pineal region, PTPR ) 樣品，進行 Agilent SureSelect RNA exome。
AB-like tumor 的 RNA exome 分析
為了同時掌握腫瘤基因表現狀況並偵測任何可能的基因突變，透過 SureSelect 外顯子系統配合 RNA 分析後，31 個樣品資料經比對，快速找出了 1793 個重要突變及基因變化 ( Fig 1c、Fig 2 )，發現與腦腫瘤相關的特定基因過表現狀況 ( overexpression )、RNA fusion、Indel 突變等等的基因異常現象 ( Fig 2 )。
並且，研究發現女性患者的 AB-like 腫瘤中，與 X 染色體相關的融合基因出現率高達 5 倍多 ( Tab 2 )，幾乎在 MN1-BEND2 融合型及 BRAFV600E 突變型的癌症樣品中都有發現，而女性患者的 22 號染色體融合發生率更高。
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Figure 1c. Astroblastoma and neural stem/progenitor cell morphologies and AB-like tumor molecular subtypes. Unsupervised hierarchical clustering of RNAseq expression data separates AB-like tumors into MN1-rearranged and MAPK/PI3K pathway transcriptomic groups. Genes overexpressed in MN1-BEND2 tumors are highlighted in red, those overexpressed in MAPK-ABC tumors in blue and PTPR in bronze. Some genes highly expressed in both MN1-BEND2 tumors and PTPR are depicted in red. |
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Figure 2. Gene mutations and fusions segregate AB-like tumor genetic groups. Pathway gene alterations across AB-like tumors, ATAB, PTPR and RELA STE are grouped. Missense = MS, splice region variant = SV, stop gained = SG, stop lost = SL, frameshift = FS, deletion = DEL, insertion = INS, fusion = FU, PT = PTPR. Detected genetic alterations are highlighted in red. Brown cells indicate fusions detected by only one read. Pink indicates a FathmmMLK score <0.5, but predicted to be damaging by PolyPhen or deleterious by SIFT. Source data: Supplementary Data 1 and 2. |
AB-like tumor 基因變異與患者年齡的相關性
該團隊發現許多 RNA fusion 或 mutation 發生在特定年齡層，例如：ABCC1 mutations/fusions 都發生在 16 歲以下患者 ( Fig 4g, 4h )、VEZT/VEZF mutations 及 CTD-2152M20.2-GOLPH3 fusions 則在 25 歲以下患者的腫瘤中 ( Fig 4g, 4h )、MEG8 fusions 大多數發生不超過 30 歲的女性，大多在 16 歲以下 ( Fig 4g, 4h ) 等等…，這些基因訊息的整合可以幫助罕見癌症 — 星形芽細胞瘤的了解，加以應用於診斷分類 ( Fig 8 )。
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Figure 4. Astroblastoma-like tumor subtypes exhibit characteristic histologies and genetic alterations. f, g Mutations in AB-like tumor related genes occur in a patient age dependent manner. ABCC1 mutations and fusions all occurred in patients 16 years old or less. Its highest expression in the Allen Human Developmental Transcriptome (AHDT) was before 25 pcw. VEZT and VEZF mutations and CTD-2152M20.2-GOLPH3 fusions were all found in tumors from patients aged 25 and younger (Supplementary Data 2). The highest GOLPH3, VEZT and VEZF1 expression in the AHDT was under 25 pcw. MEG8 fusions occurred only in females 30 years of age and younger, most below 16 years, and only in MN1-BEND2 and MAPK-A tumors. GRIA2 mutations were present in MAPK-ABC cases. ANK3, PARP8 and PTEN alterations were mostly present in non-BRAFV600E MAPK-AB patients. A PARP8 fusion was present in a BRAFV600E tumor. Conversely, high GRIA2 expression spans the fetal and postnatal AHDT, but not below approximately 12 pcw. GRIA2 mutations are absent in MN1-rearranged tumors. Some genetic lesions were relatively specific to AB-like tumor genetic types: MN1-BEND2 fusions and ABCC1 mutation or fusion in MN1-rearranged tumors, and TRIO and KALRN fusions in BRAFV600E tumors. SON mutations span the gamut of patient ages, as does its expression in the AHDT. These presumed somatic mutations were found in tumors clinically presenting at the indicated ages, however this data only indicates that the mutation was acquired in a patient sometime prior to that age, and some could have indeed been germline. Colors are arbitrary in f. In g MN1-BEND2 tumor mutated genes are in red and MAPK-ABC tumor mutated genes are in blue. Source data: Supplementary Data 1 and 2. |
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| Figure 8. Astroblastoma-like tumors show neural stem cell type-specific transcriptional lineage of developmentaly early (vRG) to later (oRG and tRG) radial glia and clinically present in comensurate sequencial patient ages. |
雖然在早期癌細胞研究中就知道 RNA fusion 具有重要意義，但過去的技術限制了臨床 FFPE 樣品的搜尋範圍，使得以往 FFPE 樣品只能做 DNA 突變分析，無法進行 RNA fusion 偵測，因此無法有效的大規模尋找不同癌種、不同病程的 RNA fusion 現象。此篇文獻的結果可看到，FFPE RNA exome 除了可以計算基因表現外，也可以搜尋 RNA fusion 現象。當您在思考如何讓 Cancer WES 計畫具有更好的剖析深度時，FFPE RNA exome 將會是您的最佳選擇。
1. Mertens F., Johansson B., Fioretos T., Mitelman F. The emerging complexity of gene fusions in cancer. Nat. Rev. Cancer. 2015;15:371–381. doi: 10.1038/nrc3947.
2. Mitelman, F., Johansson, B. & Mertens, F. The impact of translocations and gene fusions on cancer causation. Nat. Rev. Cancer 7, 233–245 (2007).
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