傳統性鋸齒狀腺瘤的臨床病理特徵及分子路徑分析

Abstract

傳統鋸齒狀腺瘤(traditional serrated adenoma, TSA)是一種獨特的鋸齒狀息肉，也是導致大腸直腸癌(colorectal carcinoma, CRC)的一種前驅腫瘤。因為TSA是大腸最少見的息肉，至今其致癌機制並不甚清楚。TSA和另一種鋸齒狀息肉:無莖性鋸齒狀腺瘤(sessile serrated adenoma, SSA)有著密切的病理關係；但是不同於SSA，TSA較常發生在大腸左側，具有乳突狀的生長結構。更重要地， TSA並非微衛星不穩定大腸直腸癌(microsatellite unstable colorectal carcinoma, MSI-H CRC)的原發腺瘤。因此，我們假設TSA的致癌路線(the traditional serrated pathway)是不同於無莖性鋸齒狀腺瘤路線(the sessile serrated pathway)。此篇研究著重於探討the traditional serrated pathway的分子病理特徵，並比較和sessile serrated pathway的差異性。 首先我們整理TSA的臨床病理特徵。我們收集過去台大醫院曾經診斷TSA的111個病人，並且回溯分析這些病人所有大腸息肉。其中46人除了TSA外，還診斷過50個增生性息肉(hyperplastic polyps, HPs), 14個SSAs，另外的27個TSAs，及17個CRCs。顯微鏡下，27%的 TSAs周圍存在SSA 或 HP，可視為TSA的前驅病變。這些有鋸齒前驅病變的TSA強烈與BRAF 基因突變相關 (P < 0.001)。另外，有BRAF突變的TSA也較常好發有其它鋸齒狀息肉，且較常在右側大腸。我們分析17個CRCs的分子病理特徵，發現較易發生於女性，具有高比例BRAF 突變(59%),59%帶有陽性CpG 島甲基化表型(positive CpG island methylator phenotype), 且77%為微衛星穩定大腸直腸癌(stable or low-levels of microsatellite instability, MSS CRC)。 這些資料顯示，診斷過TSA的病人所發生的大腸直腸癌的分子病理特性和傳統大腸直腸癌的分子特徵有很大的差異性。 我們接著分析60個有惡性轉變TSAs的分子病理特徵。病理觀察下可分為(1)16個鋸齒狀病變的TSAs (TSA-S),(2)25個傳統型腺瘤病變TSAs (TSA-C),及(3)19個管狀絨毛狀腺瘤(tubulovillous adenoma, TVA)有局部性鋸齒狀變化(TVA-S)。此三類惡性轉變TSAs具有類似比例的陽性CpG 島甲基化表型。且60個惡性轉變TSA皆保留有4種錯誤配對修復蛋白(mismatch-repair proteins)的核免疫染色，這證實這些腫瘤皆為MSS CRCs。具有高度分化不良及侵襲性癌的TSA才有P53蛋白過量表現。且TSA-S和BRAF 突變有高度相關，而TSA-C 和TVA-S則較和KRAS 基因突變有相關(P < .0001)。另外，β-catenin蛋白的核染色只發生在TSA-C和TVA-S，而非TSA-S。我們也觀察到28個從TSA轉變的大腸直腸癌，形態學常具有鋸齒狀變化，豐富嗜酸性染色細胞質、不規則浸潤的腫瘤邊緣，少數帶有大量壞死，且常具有黏液性分化。 因為最近發現壓抑Wnt/β-catenin訊息路線的環指蛋白43基因 (ring finger protein 43, RNF43)和PTEN 基因 (Phosphatase and tensin homologue deleted on chromosome 10)，在MSI-H CRC有高比例的突變，我們也分析RNF43 和PTEN於traditional serrated pathway的致病關係為何。 基因定序分析發現: 10%(2/20) SSAs, 28%(10/36) TSAs, 19% (7/37)惡性轉變的TSAs, 以及29% (9/31) BRAF突變且微衛星穩定大腸直腸癌(BRAF-mut/MSS CRCs)具有RNF43的突變；另外30個TVA/VAs並沒有發現突變(0%)。所有突變位點皆隨意分布在RNF43蛋白的環指蛋白結構域(ring finger domain of RNF43)的上游，且不具有微衛星不穩定大腸直腸癌群聚於微衛星小島的現象。另外RNF43基因突變顯著和BRAF 基因突變相關，而和KRAS無相關性 (P = .018 and .045, respectively)。 在PTEN分析中，我們先用免疫蛋白染色偵測PTEN蛋白的表現。結果發現55%惡性轉變的TSAs及57% BRAF-mut/MSS CRCs具有減弱或喪失PTEN蛋白表現(P = 0.501)。在44不正常PTEN蛋白表現的檢體中，8個有PTEN 基因突變，12個有PTEN 啟動子過度甲基化，及14個有大片段基因刪除。病理分析下，TSA惡性轉變的大腸直腸癌和BRAF-mut/MSS CRCs有相似的病理特徵。我們用Makinen’s criteria分析發現兩者都有高比例(90%)的鋸齒狀型態，包括上皮細胞鋸齒狀變化、豐富嗜酸性染色細胞質、不規則浸潤的腫瘤邊緣及常具有黏液性分化。 總結來說，診斷有BRAF突變TSA的病人傾向再發生其他鋸齒狀息肉及特殊分子特徵的大腸直腸癌，BRAF突變TSA在形態學上亦有鋸齒狀息肉的前驅病變。惡性轉變過程中，BRAF突變TSA較會走向鋸齒狀病變(serrated dysplasia)，而 KRAS突變TSA較會有傳統腺瘤病變。高度分化不良及有侵襲性癌的TSA會有過度p53蛋白表現，且皆屬於微衛星穩定大腸直腸癌。相對於傳統性管絨毛狀或絨毛狀腺瘤不具有RNF43 突變，我們發現約1/3的TSA及BRAF-mut/MSS CRCs有RNF43 突變，此突變率顯著地比SSA高。且約一半TSA惡性轉變的大腸直腸癌及BRAF-mut/MSS CRCs有PTEN蛋白異常。 我們發現TSA是一種獨特且有多元病理及分子特徵的大腸直腸腺瘤，其分子病理表現皆異於SSA。病理觀察下，TSA可直接癌化為微衛星穩定大腸直腸癌，其具有特殊的RNF43及PTEN基因突變。它和BRAF-mut/MSS CRCs有相似的型態學及分子變化，這證明這兩個腫瘤屬於同一路線的癌變過程範圍。基於以上觀察，我們定義由TSA癌變的分子路線應獨立於sessile serrated pathway，且應命名為traditional serrated pathway。

Traditional serrated adenoma (TSA) is a distinct type of serrated polyp and a precursor of colorectal cancer (CRC). Because of its rarity, the pathogenesis of TSA and TSA with neoplastic progression toward CRC is unknown. To better clarify the morphologic and molecular characteristics of the TSA pathway, we evaluated the clinicopathological and molecular features of the TSA pathway and compared with the sessile serrated (SSA) pathway by analyzing RNF43, an E3 ligase that suppresses the Wnt/β-catenin signaling pathway, and PTEN. Both genes are frequently mutated in MSI-H CRC. We first recruited 111 patients with an index TSA and analyzed the pathologic and molecular features of their synchronous/metachronous serrated neoplasms. The results showed that 46 patients had additional 50 hyperplastic polyps (HPs), 14SSAs, 27 TSAs and 17 CRCs. Twenty-seven percent of TSAs exhibited a precursor serrated polyp (SSA or HP) in the periphery and were strongly correlated with BRAF mutation (P < 0.001). Serrated polyps occurred more commonly in patients with BRAF-mutated index TSAs but not with KRAS-mutated index TSAs. BRAF-mutated index TSAs were strongly associated with right-sided location, and BRAF mutated synchronous/metachronous serrated polyps (P = 0.005). The 17 CRCs occurred more frequently in women and were characterized by a high BRAF mutation rate (59%), positive CpG island methylator phenotype (CIMP) (59%), and stable microsatellite instability (MSS) (77%), which were distinct from the molecular features of conventional CRC. We collected 60 TSAs with cytologic dysplasia and/or invasive carcinoma and categorized as TSA with serrated dysplasia (TSA-S, n = 16, (27%)), TSA with conventional adenomatous dysplasia (TSA-C, n = 25 (42%)), and tubulovillous adenoma with focal serrated feature (TVA-S, n = 19 (31%)). The frequency of the positive CIMP was similar among the 3 categories, and immunostaining of 4 mismatch-repair proteins in the nucleus was retained. Overexpression of p53 was detected only in high-grade dysplasia and invasive components. In more, TSA-S had a significantly higher frequency of BRAF mutation than TSA-C and TVA-S (P = .006), whereas TSA-C and TVA-S had higher frequencies of KRAS mutation than TSA-S (P < .0001). Abnormal nuclear accumulation of β-catenin was detected in TSA-C and TVA-S but not in TSA-S. RNF43 mutation was found in 2 of 20 (10%) SSAs, 10 of 36 (28%) TSAs, 7 of 37 (19%) TSAs with cytologic dysplasia, and 9 of 31 (29%) BRAF-mutated/microsatellite stable CRCs (BRAF-mut/MSS CRCs); however, no RNF43 mutation was found in 30 tubulovillous/villous adenomas. All mutations were located upstream of the ring finger domain of RNF43 without clustering, which is distinct from the pattern described for MSI-H CRC. RNF43 mutation was closely associated with BRAF mutation but inversely associated with KRAS mutation (P = .018 and .045, respectively). In more, aberrant PTEN expression was detected in 14 (52%) of CRCs arising from TSA (TSA-CRCs) and 30 (57%) BRAF-mut/MSS CRCs (P = 0.501). Among the 44 samples with aberrant PTEN expression, 8 harbored PTEN somatic mutations, which were characterized by random distribution without hotspot clustering, 12 had promoter hypermethylation, and 14 had deleted alleles. Most cases (>90%) of TSA-CRCs and BRAF-mut/MSS CRCs exhibited a constellation of serrated morphology, including epithelial serrations, abundant eosinophilic cytoplasm, and discernible/vesicular nuclei. In conclusion, BRAF-mutated TSA tend to exhibit a precursor serrated polyp in the periphery and be predisposed to subsequent serrated neoplasia. TSA with serrated dysplasia was associated with BRAF mutation. Most CRCs arising from TSA were MSS and had p53 nuclear accumulation. The finding of common RNF43 and PTEN mutation and similar serrated morphology in TSA-CRCs and BRAF-mut/MSS CRCs, and absent RNF43 mutation in tubulovillous/villous adenoma, indicated specific molecular features in the TSA pathway. Based on the above features, we coined TSA carcinogenesis as the TSA pathway, which should be distinguished from the SSA pathway.