Annexin A10蛋白表現於腹腔前腸腫瘤及遠處轉移之探討

Annexin A10 Protein Expression in Neoplasms and Distant Metastases of Abdominal Foregut Organs

呂庶熙(Su-Hsi Lue)

指導教授 ： 許輝吉

國立臺灣大學/醫學院/病理學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.10014

摘要

Annexin家族是有核細胞結合鈣離子而欠缺EF hands的最大一類蛋白，在人類發現有12種不同的Annexin成員。Annexin A10 (ANXA10)有數項特別表徵，包括罕見表現，以及第三重複序列的codon 與四重核心重複序列第2型鈣離子結合區的不尋常丟失。我們之前的研究顯示ANXA10有一個序列較短的變異型，稱為ANXA10S，其結構與表現均非常獨特，僅表現於正常的肝細胞或肝細胞起源的腫瘤細胞。相較於ANXA10S，ANXA10可表現於胃，但在人類的癌症中所扮演的角色則完全不清楚。
本研究主要是觀察ANXA10在正常組織的分佈與各種癌症中的表現，探討其組織與腫瘤特異性，特別是診斷潛力，故著重在觀察由腹腔前腸源起的器官在癌化及遠處轉移的表現，藉此了解ANXA10在鑑別診斷上的價值。

關鍵字：胃癌，腹部前腸，膜聯蛋白，癌症，轉移

方法：本研究主要藉由使用特殊的抗ANXA10抗體，以免疫組織化學染色法(immunohistochemical stain, IHC)，研究分析1,545個手術切除檢體。包括了胃癌(405例)、十二指腸癌(25例)、胰臟癌(73例)、總膽管癌(41例)、膽囊癌(29例)、肝癌(54例)、大腸癌(175例)、卵巢癌(35例)、其他癌症(284例)，另有218個轉移檢體，128例良性腫瘤。

結果：
(1) 在人類的成人或胚胎組織，ANXA10 "僅”大量表現在正常胃粘膜及十二指腸的Brunner氏腺體上皮的細胞核中。對於不同物種的老鼠、土撥鼠及新出生的迷你豬，ANXA10的表現在也嚴格局限於正常胃粘膜上皮。
(2) 對於癌症而言，ANXA10常表現在源起於腹腔前腸器官的腺癌。包括胃癌(57%, 231/405)、十二指腸癌(72%, 18/25)、胰臟癌(82%, 60/73)、總膽管癌(56%, 23/41)、膽囊癌(34%, 10/29)、肝內膽管癌(22%, 12/54)。
(3) 對於不是由腹腔前腸所發育而來的器官腫瘤，ANXA10也會表現在少數特定的腫瘤，包括卵巢的粘液性腫瘤，某些粘液性大腸癌及胃腸道的畸型瘤 (hamartomas)
(4) 在387例原發性胃癌(primary GC)中，ANXA10表現主要是瀰漫型(diffuse type；74%，89/120)及混合型(mix type；75%，21/28)，顯著高於腸型胃癌(intestinal type；18%，43/235)，P<1x10-8。在早期胃癌中也有相同的發現，分別是瀰漫型82%(27/33)與腸型16% (6/37)，P<1x10-8。但不論在何型的胃癌，ANXA10的表現與淋巴轉移及存活率並無明顯的相關性。
(5) 在34病患的殘存胃發生的原發性胃癌中，有83%主要的組織型態是腸型胃癌。在15例接受第二次胃半全切除手術的檢體中，有14例屬腸型胃癌(93%)，其中6例有ANXA10表現(40%)，同時較小的瘤腫(小於3公分)較常有ANXA的表現。P<0.05。
(6) 為了探討ANXA10的診斷價值，我們分析了肝及卵巢中的轉移性腫瘤，前三位分別是大腸直腸癌、胃癌及胰臟癌。相對於其中有87%大腸直腸癌及97%的胰臟癌轉移到肝臟，胃癌有較多的病例轉移到卵巢(54%), P<1x10-8。就女性患者而言，胃癌更明顯地轉移到卵巢(79%)，顯著高於大腸直腸癌(31%)、胰臟癌(11%)及乳癌(27%)，P值分別為 P<1x10-8，P<1x10-8及P <0.00003。
大部份轉移到卵巢的胃癌型態是瀰漫型/混合型(94%)，而轉移到肝的胃癌則為腸型(100%)，差異極端明顯，P<1x10-8。發生卵巢轉移的胃癌，其原發的胃癌及卵巢轉移病灶，ANXA10的表現率都很高，分別為83%及87%，都顯著高於轉移到肝臟的胃癌，分別為12%及12%，P值分別為P=0.000002 及 P=0.00002。
(7) 轉移到卵巢的兩個主要癌症是胃癌與大腸直腸癌，轉移性胃癌有87%的檢體有ANXA10表現，顯著高於轉移性大腸直腸癌(8%)，P<1x10-8。
(8) 為了探討ANXA10在原發性與轉移性卵巢腫瘤的鑑別診斷價值，我們檢驗不同型態的卵巢原發性癌症，發現ANXA10常表現在粘液性的腫瘤(囊性腺瘤為73%及囊性腺癌為61%)，而完全不表現在漿液性的腫瘤(腺瘤及腺癌)，P值分別為 P=0.0034 及 P=0.0003。
(9) 在膽管癌(cholangiocarcinoma)的兩種前期病變(precursor lesion)中，有90%(60/67)的膽道上皮贅瘤(BilIN)有ANXA10表現，顯著高於導管內乳頭狀贅瘤(IPN-B)的56% (19/33)或是腺癌(adenocarcinoma)的48% (101/210)，P<0.0006及P<1x10-8。
在284例膽道系統的癌症中，ANXA10較常表現在低位的膽道如胰臟或壺腹(ampulla of Vater)，高於總膽管、膽囊及肝臟，比例分別為77% (76/99)比31% (40/127), P<1x10-8。
(10) 在肝門區或是發生在較大膽管之膽管癌的ANXA10表現，顯著高於肝內發生的週邊膽管癌(43%比9%, P<0.01)，同時，分化較好的膽管癌的ANXA10表現(38%) 也會顯著高於分化較差者(3%)，P=0.0008。
(11) 在73例胰臟的腺癌中有78%有ANXA10表現，這是所有腹腔前腸腫瘤中ANXA10表現最高者，可能與胰臟癌多為分化型有關，而分化較好的胰臟癌的ANXA10表現也確實顯著高於分化較差者(87%比50%，P=0.002)。ANXA10也常表現在胰臟的粘液性腫瘤(88%，7/8)，而漿液性腫瘤則完全不表現(0%，0/13)，P<0.0001。

結論：
(1) ANXA10蛋白是一核蛋白且呈現組織與細胞特異性表現，僅表現於正常胃粘膜上皮的細胞，及十二指腸的Brunner氏腺體，其特異性表現與消化道胃粘膜的發生與性狀的維繫有絕對的關係。
(2) ANXA10在腫瘤的表現也呈現侷限性，由腹腔前腸發育而來的器官的腺癌常見ANXA10的表現，ANXA10罕見表現於其他組織器官，也不會表現於非簡單柱狀黏液細胞。故有胃黏膜上皮細胞特異的ANXA10表現時可視為腫瘤胃化生(metaplasia)與分化(differentiation) 的表徵。
(3) 胃癌的ANXA10表現與組織學型態有重要的相關性，主要表現在仍保留胃粘膜特性的瀰漫型及混合型，較不表現在腸型。此一結果與胃癌的組織發生學，及ANXA10與正常胃粘膜之相關性具一致性。不過在本研究中ANXA10的表現與否和胃癌的分期、淋巴轉移及預後並無顯著的相關性。
(4) 因良性胃疾而全胃切除之殘胃，二十餘年後發生的原發性胃癌，組織學型態主要是腸型。而在這些腸型的胃癌ANXA10多表現小於3公分的腫瘤中。
(5) 與其他主要癌症相比，胃癌更常轉移至卵巢。轉移到卵巢的胃癌組織學型態多為瀰漫型/混合型，不論是原發部位的胃癌或卵巢的轉移部位的組織大部份都有ANXA10的表現。反之，大部份轉移到肝臟的胃癌細胞組織學型態是腸型，不論是原發部位或轉移部位的胃癌組織都很少有ANXA10的表現。
(6) 與胃癌相比，轉移到卵巢的大腸直腸癌的ANXA10表現率極低，所以ANXA10表現可以用來做兩者的鑑別診斷。
(7) ANXA10在卵巢的良性或惡性之粘液性腫瘤表現率高，而漿液性腫瘤則罕見，ANXA10的表現有助鑑別診斷；在胰臟中亦有相同的現象，表示卵巢與胰臟粘液性腺瘤與腺癌都常見胃型化生或分化，有助於腫瘤發生的探討。但就卵巢的原發性與轉移性粘液性腺癌，單獨ANXA10的表現無法當成鑑別診斷的指標(marker)。
(8) ANXA10在BilIN、IPN-B及膽道的腺癌之表現率都相當高，表示在膽道系統的腺癌在早期癌化過程中，導管上皮細胞可能存在著普遍的胃型化生。
(9) 在肝臟的膽管癌中，位於肝門區且分化較好的膽管癌有最高的ANXA10表現率，而發生在肝週邊且分化較差的膽管癌ANXA10的表現率幾近於零，P<0.0004。顯示ANXA10與膽管癌的細胞分化及發生在肝中膽管位置有相關性。
(10)在胰臟的腺癌中，ANXA10的表現也與癌細胞的分化有相關性。
基於以上的發現，本研究提出以下的論點:
(1) ANXA10是一個對胃粘膜上皮細胞具有專一性的指標，亦是細胞發生胃型化生的指標，常見表現於腹腔前腸來源器官腺癌，具有鑑別診斷的價值，也有助於特定部位之簡單立方上皮的良性或惡性腫瘤發生之探討。
(2) ANXA10對於卵巢發生的胃癌或大腸直腸癌轉移是一個有用的鑑別診斷指標。就胃癌而言，ANXA10的表現與否與胃癌組織型態及肝臟或卵巢轉移傾向有極高的相關性，此一特殊現象的意含與轉移機制和AXNA10表現的關聯性有待進一步探討。同時是否對其他器官的遠處轉移也可能具有適用性，特別是肺、腦與骨骼，亦有待研究。

關鍵字

胃癌 ； 腹部前腸 ； 膜聯蛋白 ； 癌症 ； 轉移

並列摘要

Annexins (ANXs) belong to the largest single category of eukaryotic calcium-binding proteins without EF hands, and there are 12 human annexin members. ANXA10 has several distinct features, including its rare expression, codon deletion in the conserved repeat 3, and an unusual ablation of the type II calcium-binding sites in tetrad core repeats 1, 3 and 4. Our previous study showed that ANXA10 had a novel, unique short isoform (ANXA10S), initially mistaken as ANXA10 (Liu et al., 2002), which was expressed exclusively in liver cells and neoplasms of liver cell lineage. ANXA10 was detected in the stomach but its tissue distribution and role in human cancer are not known. This study was aimed to determine the expression pattern of ANXA10 protein in normal tissues and human cancers, with emphasis on its expression in neoplasms of the organs derived from the abdominal foregut and the differential diagnostic value in the major metastases of the liver and ovary.
and to explore its diagnostic potential.

Key words: annexin, gastric carcinoma, abdominal foregut, cancer, metastasis

Methods: The immunohistochemical stain (IHC) using a specific rabbit polyclonal antibody against ANXA10 protein was applied to examine the tissue distribution of ANXA10 protein in various types of human neoplasms. A total of 1,545 surgical samples of primary and metastatic tumors were analyzed, including neoplasms of the stomach (405 cases), duodenum (25 cases), pancreas (73 cases); common bile ducts (41 cases); gallbladders (29 cases); liver (54 cases); colon (175 cases); ovary (35 cases); and other organs (284 cases), such as brain, thyroid, breast, oral cavity, pharynx, larynx, lung, esophagus, kidney, urinary bladder, testis, uterus and cervix, malignant lymphoma. The metastatic tumors of the liver and ovary included 218 of metastastic carcinomas and 128 benign tumors.

Results:
(1) ANXA10 protein was located in the nucleus and expressed diffusely but “exclusively” in the epithelial cells of normal fetal and adult human gastric mucosa and Brunner’s gland of the duodenum.
(2) ANXA10 was detected in various types of adenocarcinomas of the organs derived from the abdominal foregut, including the stomach (57%, 231/405), duodenum (72%, 18/25), pancreas (82%, 60/73), common bile duct (56%, 23/41), gall bladder(34%, 10/29), cholangiocarcinoma (22%, 12/54).
(3) In organs other than those derived from the abdominal foregut, ANXA10 protein was expressed in a limited number of neoplasms, including mucinous tumors of the ovary, some mucinous carcinomas of the colon, and hamartomas of the GI tract.
(4) In 387 primary gastric cancer (GC), ANXA10 was detected most commonly in diffuse type GC (DGC; 74%, 89/120) and mixed type GC (MGC; 75%, 21/28), significantly higher than the intestinal type (IGC; 18%, 43/235), P<1x10-8. In early GC, the expression ANXA10 was also found more frequently in DGC/MGC than IGC, 82% versus 16%, P<1x10-8.
(5) ANXA10 expression in gastric cancer did not correlate with lymph node metastasis and patient’s outcome in both DGC/MGC and IGC.
(6) The major histological type of the 34 remnant GC were IGC (83%). In 15 resected advanced remnant GC cases, 93% were IGC, 6 were positive for ANXA10. ANXA10 protein was frequently expressed in the tumors which size is less than 3cm, P<0.05.
(7) The great majority of the colorectal (87%) and pancreatic cancers (97%) metastasized to the liver, whereas the gastric carcinoma metastasized more frequently to the ovary than the colorectal and pancreatic cancers, P<1x10-8.
In female patients, gastric carcinoma predominantly metastasized to the ovary (79%), significantly higher than those of the colorectal cancer (31%), pancreatic cancer (11%) and breast cancer (27%), P<1x10-8 , P<1x10-8 and P <0.00003, respectively..
The great majority of ovarian metastases of GCs were DGC/MGC (94%), whereas the liver metastases were predominantly IGC (100%), P<1x10-8.
(8) As compared with metastatic colorectal cancer, ANXA10 was detected in 87% of 31 metastatic gastric cancers to the ovaries, but only in 8% of the 39 colorectal cancer, P<1x10-8
(9) ANXA10 protein was frequently expressed in mucinous tumors of the ovary tumors (73% in cystadenomas and 61% in cystadenocarcinomas), but in none of serous adenomas and adenocarcinomas, P=0.0034 and P=0.0003, respectively.
(10) ANXA10 was detected in 90% (60/67) of the BilIN lesions, significantly higher than those of the IPN-B lesions (56%, or 19/33) and adenocarcinoma (48%, or 101/210), P<0.0006 and P<1x10-8, respectively.
(11) A pair-wise analysis showed the differentiated cholangiocarcinoma at the hilar region had the highest positive rate for ANXA10 protein expression (63%), whereas the poorly differentiated cholangiocarcinomas of the liver parenchyma the lowest (0%), P<0.0004
(12) ANXA10 in pancreatic cancers was also expressed more frequently in well to moderately differentiated tumors than the poorly differentiated and undifferentiated tumors 87% versus 50%, P=0.0021.

CONCLUSION:
(1) ANXA10 was diffusely expressed in the nuclei of normal gastric mucosa, but absent or significantly decreased in the intestinal metaplasia.
(2)ANXA10 was detected in adenocarcinomas of organs derived from the abdominal foregut.
(3) ANXA10 might play a role in the maintenance of gastric differentiation. However, the ANXA10 expression did not correlate with tumor stage, lymph node metastasis and patient’s outcome.
(4) Consistent with correlation with histological types of GC, the expression of ANXA10 in the remnant GC, in which the IGC predominated, was low.
(5)The great majority of ovarian metastases of GCs were diffuse/mixed type GCs, and hence most of the primary GCs and ovarian metastases were positive for ANXA10 expression, whereas the liver metastases were predominantly IGCs, and mostly negative for ANAX10 expression.
(6)ANXA10 protein expression, which was commonly positive in the metastatic gastric carcinoma to the ovaries, but rarely in the metastatic colorectal carcinoma (CRC), could serve as a diagnostic aid.
(7) The most of the ovarian mucinous neoplasms showed gastric metaplasia or differentiation, and ANXA10 per se was not a differential diagnostic marker.
(8) The high positive rate of ANXA10 expression in the BilIN, IPN-B, and biliary tree adenocarcinoma suggest that gastric metaplasia is an early and common event in the multistep malignant transformation of biliary adenocarcinoma.
(9) The expression of ANXA10 in cholangiocarcinoma correlated with tumor cell differentiation and the level of intrahepatic bile duct.
(10) In pancreas adenocarcinoma, the ANXA10 protein expression was also correlated with differentiated tumors.

Based on the above findings, we suggest that:
(1) ANXA10 is a specific biomarker for gastric mucosal epithelial cells, and a marker for gastric metaplasia or differentiation in benign and malignant neoplasms of the abdominal foregut and adenocarcinoma with simple columnar epithelium in several other anatomic sites.
(2) With certain caution, ANXA10 is a useful differential biomarker for the metastatic gastric and colorectal cancers of the ovaries, and probably other distant metastases.

並列關鍵字

annexin ； gastric carcinoma ； abdominal foregut ； cancer ； metastasis

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