胰腺实性假乳头状肿瘤的组织发生及Notch信号通路的表达研究
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摘要
胰腺实性假乳头状肿瘤(solid-pseudopapillary neoplasm,SPN)是一种少见的具有低度恶性潜能的实体性肿瘤,好发于年轻女性,约占胰腺肿瘤的1-2%。过去因对其认识不足,易误诊为无功能胰岛细胞瘤,胰腺囊腺瘤,乳头状腺泡细胞癌等,但其预后较胰腺导管腺癌及胰腺内分泌肿瘤要好。
近年来因对胰腺实性假乳头状肿瘤的病理形态学认识的提高以及新的免疫组化标记的广泛应用,国内外对SPN的文献报道病例明显增多,但对其组织起源及发病机理的认识尚不清楚,对其组织起源存在多种假设,有认为SPN可能起源于胰腺导管上皮细胞,有的认为SPN可能起源于胰腺腺泡细胞,有的认为来自胰腺内分泌细胞或胚胎神经嵴,也有人认为可能起源于胰腺多潜能干细胞,但目前尚无一致的结论,对胰腺SPN的组织发生的探讨是SPN研究的一大热点。
2001年Tanaka等报道胰腺实性假乳头状肿瘤几乎所有病例均存在Wnt-p-catenin信号通路的活化,即编码β-catenin的CTNNB1基因的3号外显子出现点突变,导致β-catenin蛋白难以在肿瘤细胞胞浆内磷酸化及降解并与Tcf/Lef形成复合物进入细胞核内,激活MYC及Cyclin D1等癌基因的转录,即Wnt-β-catenin信号通路的活化。免疫组化染色β-catenin蛋白由正常胰腺导管及腺泡细胞胞膜阳性转而在SPN肿瘤细胞呈胞浆及胞核阳性,这一染色特点对病理学诊断SPN有重要意义。但除Wnt-β-catenin信号通路以外,有无存在其它信号传导通路的改变,有无其它癌基因或抑癌基因在SPN的发生发展中起作用,近年来有关这方面的研究国内外极少有文献报道。
DNA拷贝数的变化在人类遗传性疾病及肿瘤等的发生发展中起重要作用,基于微阵列技术的比较基因组杂交(aCGH)通过在一张芯片上用标记不同荧光元素的样品(肿瘤样品和对照样品)同时进行杂交,可检测样本基因组和对照基因组之间DNA拷贝数的变化,直观地表现出肿瘤及遗传性疾病基因组DNA在整个染色体组的缺失或扩增,对肿瘤而言缺失部位可能包含抑癌基因,而扩增片段则可能存在癌基因。这对于寻找与肿瘤发生相关的癌基因及抑癌基因非常有帮助,通过对SPN标本进行aCGH检测,捕获与SPN发生相关的基因进行深入的研究,对于了解SPN的发生及发展有重要意义。
2003年1月至2010年6月在浙江大学医学院附属第一医院就诊并经穿刺及手术切除病理诊断为SPN者共48例,为探讨SPN的组织起源我们选择病史资料完整,有随访结果及有足够肿瘤组织的SPN病例共20例进行形态学,免疫组织化学及超微结构观察,并选择同期因胰腺导管腺癌及胰腺神经内分泌肿瘤行手术切除的标本各15例作免疫组化对照,选择目前诊断SPN特异的免疫组化标记物β-catenin、E-cadherin,胰腺导管上皮标记物CKpan胰腺腺泡细胞标记物α-1-AT胚胎神经嵴的标记物S-100蛋白,Melan A神经内分泌细胞标CgA、Syn,CD56及Secretagogin蛋白等抗体,进行免疫组化检测,结果发现,本组SPN91%为女性,平均年龄为33.4岁,肿瘤大小1.2～14CM,切面易见出血及囊性变,组织学上由粘附性差的单一的上皮样细胞形成实性及假乳头状结构,超微结构肿瘤细胞胞浆内有丰富的细胞器,主要为肿胀的线粒体及酶原样颗粒,未见神经内分泌颗粒。免疫组化所有SPN肿瘤细胞β-catenin胞浆及胞核阳性,β-catenin胞膜阴性,E-cadherin免疫组化染色显示胞膜染色完全丧失。80%SPN肿瘤细胞表达CKpan,但其染色强度与正常胰腺上皮细胞相比要弱一些,着色细胞数量与正常胰腺上皮细胞相比也要少。95%SPN肿瘤细胞表达α-1-AT,但呈小簇状及单个细胞阳性。100%肿瘤细胞CD56胞膜阳性,55%肿瘤细胞Syn胞浆阳性,但特异性的神经内分泌标记物CgA只有5%肿瘤细胞胞浆阳性,Secretagogin肿瘤细胞均阴性,S-100有1例(5%),Melan A有2例(10%)肿瘤细胞胞浆阳性,我们的形态学及免疫组化观察结果支持SPN起源于胰腺多潜能干细胞,并向胰腺外分泌,内分泌及神经外胚叶多向分化,其不同的免疫组化标记表达可能是多潜能干细胞沿不同方向或不同阶段分化的结果。
为了解在SPN发生及发展过程中除Wnt-β-catenin信号通路活化外,有无其它癌基因,抑癌基因及其相关信号通路的改变,我们在检测β-catenin基因3号外显子突变的基础上,对10例SPN样本进行了高通量的aCGH检测,若将待测DNA与对照DNA荧光强度之比的log2 ratio≥0.2或≤-0.2,即相当于每个位点≥3个DNA拷贝数改变,分别作为DNA有扩增或缺失的阈值,结果在10例SPN样本中共检测到有DNA拷贝数改变的染色体区域112个,最多的一例有53个,最少的12个,平均每例22.3个,每一样本检测到扩增的区域要明显多于缺失的区域,平均每例检测到扩增区域19.5个,缺失区域3.4个,最常见的扩增区包括1p,2q,4q,5q,8p,9p, 10q, 11q,13q,15p,15q,16p,19q,20p,21q,22q, Xp,而缺失的区域主要位于11q,17p,20p及Y染色体上。若将Log2 ratio定于≥0.75或<-0.75,即相当于阈值3.5倍的DNA拷贝数改变作为高水平扩增或缺失阈值,则共有58个区域检测到高水平扩增,主要是lp,2q,4q,5q,9p,10q, llq,13q,15p,15q, 19q,20p,21q,22q,而缺失则位于11q及Y染色体上。对于存在高水平扩增的DNA区域,通过查找胰腺表达数据库,筛查相关的候选基因,我们发现除Wnt-β-catenin信号通路相关的几个基因位点出现扩增外,还发现Notch 2所在染色体1p12区域易检测到高水平的扩增,其次为OR4F4等所在的15q26.3及OR4M1等所在的14q11区域易检测到高水平扩增。而在胰腺导管腺癌中易出现的KRAS,P53,P16及DPC4基因及CDKN2A, SMAD4, DCC等抑癌基因未能在SPN中检测到,提示在SPN的发生及发展中出现的分子遗传学改变与胰腺导管腺癌不同。
SPN是一种具有低度恶性潜能的胰腺实体性肿瘤,目前尚无判断其预后的形态学金标准,我们对作aCGH检测的10例SPN标本,以①肿瘤>5CM；②出现肿瘤性坏死；③侵犯胰腺周围软组织；④脉管/神经周围侵犯；⑤核分裂像增多或增殖指数增高作为具有侵袭性特征的组织学指标,我们发现具有较多侵袭性特征组织学指标的SPN样本易检测到DNA拷贝数改变,且出现高水平扩增者要多于无侵袭性组织学特征的SPN样本。
在aCGH初步筛选的基础上,为进一步探讨Notch信号通路在SPN发生发展中的作用,我们选择冷冻新鲜的SPN, PDAC, PET及正常胰腺组织样本各10例,用RT-PCR法检测了Notch信号通路的主要成分Notch1, Notch2, Notch4及Jagged-1 mRNA在这些样本中的表达,并选择SPN, PDAC, PET及正常胰腺组织各20例常规石蜡存档标本,用免疫组化法检测了Notch 1, Notch3, Jagged-1及CyclinD1蛋白在这些样本中的表达情况,我们发现Notch2 mRNA在SPN中表达明显上调,Notch1, Jagged-1 mRNA在SPN中表达亦上调,Notch4 mRNA的表达未见明显改变。Notch1, Jagged-1及CyclinD1蛋白在SPN中表达明显上调,而Notch3未见明显改变,提示Notch信号通路与SPN的发生有密切的关系,活化的Notch信号通路有助于维持SPN部分肿瘤细胞处于未分化的多潜能干细胞状态。Notch信号通路可能通过CyclinD1与Wnt-β-catenin信号通路协同作用,参与SPN的发生发展。免疫组化法检测Notch1及Jagged-1在SPN中呈强阳性,而在胰腺神经内分泌肿瘤中呈弱阳性或不表达,有助于我们运用此方法在日常病理诊断中将胰腺神经内分泌肿瘤与胰腺实性假乳头状肿瘤鉴别开来。
根据我们的实验结果,可以得出以下结论：
1.胰腺SPN可能起源于胰腺多潜能干细胞，并向胰腺外分泌，内分泌及神经嵴多向分化,其不同的免疫组化标记表达可能是多潜能干细胞沿不同方向或不同阶段分化的结果。
2.aCGH检测结果提示SPN除存在Wnt-β-catenin信号通路异常活化外,可能与Notch信号通路的活化有关,其发生发展中出现的分子遗传学改变与胰腺导管腺癌不同,组织学上具有较多的侵袭性特征者,aCGH易检测出高水平扩增。
3.Notch信号通路中的主要成分Notch1, Notch2及Jagged-1在SPN中呈高表达,Notch3, Notch4弱表达或不表达,提示Notch信号通路与SPN的发生发展有密切关系。免疫组化法检测Notch1, Jagged-1有助于我们在日常病理诊断中将胰腺实性假乳头状肿瘤与胰腺神经内分泌肿瘤鉴别开来。
Solid-pseudopapillary neoplasm(SPN) is a rare and low-grade malignant neoplasm.They occur predominanlly in young women,accounting for 1%-2% of all pancreatic neoplasm.They used to be misdiagnosed as no-functional islet cell tumor,cystic adenoma and papillary acinic cell carcinoma of the pancreas in the past. But their prognosis is much better than ductal adenocarcinoma and endocrine neoplasm of the pancreas. So, it has great signicance to understand the histogenesis and differential diagnosis of solid-pseudopapillary neoplasm of the pancreas.
The reported cases of SPN have been increased greatly duo to the realization of their morphology and wide use of new immunohistochemical markers recently. But the origin and pathogenesis of SPN are not clear. There are several speculations in the histogenesis of SPN such as ductal epithelial, acinic cell, endocrine cell, embryonic neural crest or pluri potent indifferent stem cell origin. The exploration of the histogenesis of SPN is a hotpoint of study of pancreatic neoplasms.
In 2001,Tanaka et al reported that almost all solid-pseudopapillary neoplasms harbor somatic point mutation in exon 3 of CTNNB1, the gene encodingβ-catenin protein that escapes intracytoplasmic phosphorylation and subsequent degradation and that therefore binds to the T-cell transcription factor(Tcf)/Lymphoid enhancer-binding factor(Lef).Theβ-catenin-Tcf/Lef complex is then abnormally translocated to the nucleus and activates the transcription of several oncogenic genes including MYC and Cyclin D1.This leads to the activation of the Wnt/β-catenin signaling pathways. The loss of membrane expression of (3-catenin on immunohistochemistry and neuclear expression ofβ-catenin of tumor cells may be helpful in establishing the diagnosis of a solid-pseudopapillary neoplasm of the pancreas. Aside from activation of the Wnt-β-catenin pathway, little is known about other molecular pathways, oncogenes or tumor suppressor genes involved in the pathogenesis of SPN.
DNA copy number variations (CNVs) play an important role in the pathogenesis and development of human genetic diseases and tumors. In recent years, array-based comparative genomic hybridization (aCGH) has been used to analyze the CNVs of tumor samples at a genomic scale and at progressively higher resolutions. Cy3-dCTP and Cy5-dCTP fluorescent dyes were used for labeling of the tumor DNA and reference DNA respectively and were mixed and co-hybridizaed with a high-resolution DNA microarray. Copy number aberrations of large or small genomic regions can be tested between tumor samples and reference DNA. Genetic changes including amplification or loss of genomic DNA of tumors or genetic diseases can be detected at a whole chromosome level. The current concept is that frequently occurring regions of DNA amplification commonly harbor oncogenes, whereas regions of recurrent deletion harbor tumor suppressor genes. This approach may aid in screen for novel candidate oncogenes or tumor suppressor genes involved in the pathogenesis of tumors. Analysing SPN samples using aCGH to identify different types of genomic aberrations may help us to understand the pathogenesis and development of SPN.
From January 2003 to June 2010, there were 48 cases of SPN which were resected or biopsied and were pathologically confirmed in the First Affiliated Hospital of Zhejiang University. To explore the histogenesis of SPN, we selected 20 cases of SPN which had detailed history, follow-up information and were big enough for histological, immunohistochemical and ultrastructural analysis.15 cases of ductal adenocarcinoma and 15 cases of neuroendocrine tumor of pancreas were also selected as reference in immunohistochemical study. E-cadherin andβ-catenin were selected as specific SPN markers. CKpan was selected as pancreatic duct cell marker. Alpha-1-antitrypsin (α-1-AT) was selected as acinic cell marker. S-100 protein and Melan A were selected as neural crest and melanocytic markers. Chromogranin A, Synaptophysin, CD56 and Secretagogin were selected as neuroendocrine markers.91% of our cases was female patients and average age was 33.4 years. The size of our SPN cases ranged from 1.2'to 14 cm. The cut section reveals brown to yellow solid areas with haemorrage and cystic degeneration. Histologically, SPN are composed of poorly cohesive monomorphic cells forming solid and pseudopapillary structures.By electron microscope, the neoplastic cells contain abundant cytoplasm, which is rich in mitochondria, abundant rough endoplasmic reticulum and zymogen-like granules of variable size. Neurosecretory-like granules have not been found. Immunohistochemically, all SPN express nuclear/cytoplasmicβ-catenin and loss of membrane expression of E-cadherin.80% (16/20) cases of SPN neoplastic cells are positive for CKpan, but staining is of a weak intensity compared with normal pancreatic epithelium.95%(19/20) cases of SPN are positive forα-1-antitrypsin but exhibiting sparsely granular staining in the cytoplasm with a focal localization. Immunoreactivity for CD56 is 100% positive and Synaptophysin is positive in 55%cases of SPN. But the specific neuroendocrine marker Chromogranin A is positive in 5%(1/20) cases of SPN and the new neuroendocrine marker Secretagogin is negative in all cases of SPN studied. S100 protein expressed in 5%(1/20) cases of SPN and Melan A are expressed in 10%(2/20) cases of SPN. Our morphological and immunohistochemical results support the hypothesis that SPN may derived from a pluripotent indifferent stem cell capable of exocrine, endocrine and neural crest differentiation. The different expression profiles of tumor cells for immunohistochemical markers may be the results of the pluripotent indifferent stem cells differentiated at different direction or at different stage.
In order to investigate the molecular pathways, oncogenes, and tumor suppressor genes involved in the pathogenesis and development of SPN aside from activation of the Wnt-β-catenin pathway, we carried out high-resulation aCGH analysis in 10 cases of SPN. In total,112 regions of CNVs were identified in 10 SPN samples. The average number of alterations per tumor was 22.3, ranging from 12 to 53 regions per case. The frequent amplifications were detected on chromosome arms 1p,2q,4q,5q,8p,9p, 1Oq, 11q,13q,15p,15q,16p,19q,20p,21q,22q and Xp. The frequent losses were observed on chromosome arms 11q,17p,20p and chromosome Y. The threshold for genetic "gains" or "losses" was determined as log 2 ratio≧0.2 or≦-0.2 respectively. High-level amplifications or homozygous deletions were defined as a log 2 ratio≧0.75 or≦-0.75 respectively, which corresponds to a theoretical±3.5 fold of the threshold for low-level amplifications.58 regions of high-level amplifications were identified, most of which were located on 1p,2q,4q,5q,9p, 10q, 11q,13q,15p,15q,19q,20p,21q and 22q. Homozygous deletions were observed on 11q and chromosome Y. Regions of frequent high -level amplifications and homozygous deletions were screened for candidated genes using the pancreastic expression database. We found aberrant activation of the Wnt-p-catenin pathway in SPN led to increased expression of several genes associated with Wnt pathway. High -level amplifications can be detected on chromosome 1p12, where harbor Notch 2 gene,15q26.3, where contain OR4F4 and 14q11, where contain OR4M1 gene. Alterations in the KRAS, TP53, P16, DPC4 and CDKN2A, SMAD4, DCC genes, frequently found in ductal adenocarcinomas of pancreas, have not been identified in SPN. Our results suggested that the genetic alterations in the pathogenesis of SPN were different from those of ductal adenocarcinoma of the pancreas.
Solid-pseudopapillary neoplasms of the pancreas are low-grade malignant tumors, their biological behavior can not be stratified solely on the basis of histopathological criteria. Based on a literature search, we selected the following histopathological features as indicators of potentially aggressive tumor biology of our 10 cases of SPN performing aCGH analysis.(1) tumor size > 5cm.②tumor necrosis.③invasion into peripancreatic soft tissues.④lymphvascular/perineural invasion.⑤increased mitotic /proliferative rate. We found that SPN with aggressive histopathological features tend to have more DNA CNVs. High-level amplifications can be easily detected by aCGH in SPN samples with more aggressive histopathological features.
To further confirm the results obtained from our aCGH analysis and to explore the role of Notch signaling pathway in the pathogenesis and development of SPN, we selected 10 cases of frozen specimens of SPN, PDAC, PET and normal pancreatic tissues respectively for RT-PCR study. The expression of members of Notch pathway (Notch 1, Notch2, Notch4 and Jagged-1) mRNA were detected using RT-PCR. The expression of Notchl, Notch 3, Jagged-1 and CyclinDl proteins were detected in 20 cases of SPN, PDAC, PET and normal pancreatic tissues respectively using immunohistochemistry. RT-PCR revealed over-expression of Notch 2 and Jagged-1 mRNA relative to the normal pancreatic tissues. Notch 1 mRNA over-expression was confirmed in SPN, although this was also observed in PDAC.Notch 4 expression was up-regulated and this was not observed in PDAC and PET. Immunohistochemistry revealed strong positive staining of Notch 1, Jagged-1 and Cyclin D1 proteins in SPN.Notch3 protein showed weak positive staining in SPN, PET, PDAC and normal pancreatic tissues. Our findings suggested that Notch signaling pathway was involved in the pathogenesis of SPN. Activated Notch in SPN may contribute to the maintenance of undifferentiated pluripotent stem cells. Notch signaling pathway and Wnt-β-catenin pathway may be activated in SPN through CyclinDl.Strong immunostaining of Notchl and Jagged-1 in SPN may be helpful to distinguish SPN from PET in our routine pathological diagnosis.
Based on the results of our experiments, we concluded:
1. SPN may be derived from a pluripotent indifferent stem cell capable of exocrine, endocrine and neural crest differentiation. The different expression profiles of tumor cells for immunohistochemical markers may be the results of the pluripotent indifferent stem cells differentiated at different direction or at different stage.
2. Aside from activation of the Wnt-β-catenin pathway, aCGH analysis results suggested that Notch pathway was also activated in SPN. The genetic alterations in the pathogenesis of SPN were different from those of ductal adenocarcinomas of the pancreas. High-level amplifications can be easily detected in SPNs with more aggressive histopathological features.
3. Members of Notch pathway (Notch 2, Notch 1, and Jagged-1) were up-regulated in SPN. Notch 3 and Notch 4 were weakly expressed in SPN. Our results suggested that Notch signaling pathway was involved in the pathogenesis of SPN. The differential immunstaining of Notch 1 and Jagged-1 proteins may be helpful to distinguish SPN from PET in our routine pathological diagnosis.
近年来因对胰腺实性假乳头状肿瘤的病理形态学认识的提高以及新的免疫组化标记的广泛应用,国内外对SPN的文献报道病例明显增多,但对其组织起源及发病机理的认识尚不清楚,对其组织起源存在多种假设,有认为SPN可能起源于胰腺导管上皮细胞,有的认为SPN可能起源于胰腺腺泡细胞,有的认为来自胰腺内分泌细胞或胚胎神经嵴,也有人认为可能起源于胰腺多潜能干细胞,但目前尚无一致的结论,对胰腺SPN的组织发生的探讨是SPN研究的一大热点。
2001年Tanaka等报道胰腺实性假乳头状肿瘤几乎所有病例均存在Wnt-p-catenin信号通路的活化,即编码β-catenin的CTNNB1基因的3号外显子出现点突变,导致β-catenin蛋白难以在肿瘤细胞胞浆内磷酸化及降解并与Tcf/Lef形成复合物进入细胞核内,激活MYC及Cyclin D1等癌基因的转录,即Wnt-β-catenin信号通路的活化。免疫组化染色β-catenin蛋白由正常胰腺导管及腺泡细胞胞膜阳性转而在SPN肿瘤细胞呈胞浆及胞核阳性,这一染色特点对病理学诊断SPN有重要意义。但除Wnt-β-catenin信号通路以外,有无存在其它信号传导通路的改变,有无其它癌基因或抑癌基因在SPN的发生发展中起作用,近年来有关这方面的研究国内外极少有文献报道。
DNA拷贝数的变化在人类遗传性疾病及肿瘤等的发生发展中起重要作用,基于微阵列技术的比较基因组杂交(aCGH)通过在一张芯片上用标记不同荧光元素的样品(肿瘤样品和对照样品)同时进行杂交,可检测样本基因组和对照基因组之间DNA拷贝数的变化,直观地表现出肿瘤及遗传性疾病基因组DNA在整个染色体组的缺失或扩增,对肿瘤而言缺失部位可能包含抑癌基因,而扩增片段则可能存在癌基因。这对于寻找与肿瘤发生相关的癌基因及抑癌基因非常有帮助,通过对SPN标本进行aCGH检测,捕获与SPN发生相关的基因进行深入的研究,对于了解SPN的发生及发展有重要意义。
2003年1月至2010年6月在浙江大学医学院附属第一医院就诊并经穿刺及手术切除病理诊断为SPN者共48例,为探讨SPN的组织起源我们选择病史资料完整,有随访结果及有足够肿瘤组织的SPN病例共20例进行形态学,免疫组织化学及超微结构观察,并选择同期因胰腺导管腺癌及胰腺神经内分泌肿瘤行手术切除的标本各15例作免疫组化对照,选择目前诊断SPN特异的免疫组化标记物β-catenin、E-cadherin,胰腺导管上皮标记物CKpan胰腺腺泡细胞标记物α-1-AT胚胎神经嵴的标记物S-100蛋白,Melan A神经内分泌细胞标CgA、Syn,CD56及Secretagogin蛋白等抗体,进行免疫组化检测,结果发现,本组SPN91%为女性,平均年龄为33.4岁,肿瘤大小1.2～14CM,切面易见出血及囊性变,组织学上由粘附性差的单一的上皮样细胞形成实性及假乳头状结构,超微结构肿瘤细胞胞浆内有丰富的细胞器,主要为肿胀的线粒体及酶原样颗粒,未见神经内分泌颗粒。免疫组化所有SPN肿瘤细胞β-catenin胞浆及胞核阳性,β-catenin胞膜阴性,E-cadherin免疫组化染色显示胞膜染色完全丧失。80%SPN肿瘤细胞表达CKpan,但其染色强度与正常胰腺上皮细胞相比要弱一些,着色细胞数量与正常胰腺上皮细胞相比也要少。95%SPN肿瘤细胞表达α-1-AT,但呈小簇状及单个细胞阳性。100%肿瘤细胞CD56胞膜阳性,55%肿瘤细胞Syn胞浆阳性,但特异性的神经内分泌标记物CgA只有5%肿瘤细胞胞浆阳性,Secretagogin肿瘤细胞均阴性,S-100有1例(5%),Melan A有2例(10%)肿瘤细胞胞浆阳性,我们的形态学及免疫组化观察结果支持SPN起源于胰腺多潜能干细胞,并向胰腺外分泌,内分泌及神经外胚叶多向分化,其不同的免疫组化标记表达可能是多潜能干细胞沿不同方向或不同阶段分化的结果。
为了解在SPN发生及发展过程中除Wnt-β-catenin信号通路活化外,有无其它癌基因,抑癌基因及其相关信号通路的改变,我们在检测β-catenin基因3号外显子突变的基础上,对10例SPN样本进行了高通量的aCGH检测,若将待测DNA与对照DNA荧光强度之比的log2 ratio≥0.2或≤-0.2,即相当于每个位点≥3个DNA拷贝数改变,分别作为DNA有扩增或缺失的阈值,结果在10例SPN样本中共检测到有DNA拷贝数改变的染色体区域112个,最多的一例有53个,最少的12个,平均每例22.3个,每一样本检测到扩增的区域要明显多于缺失的区域,平均每例检测到扩增区域19.5个,缺失区域3.4个,最常见的扩增区包括1p,2q,4q,5q,8p,9p, 10q, 11q,13q,15p,15q,16p,19q,20p,21q,22q, Xp,而缺失的区域主要位于11q,17p,20p及Y染色体上。若将Log2 ratio定于≥0.75或<-0.75,即相当于阈值3.5倍的DNA拷贝数改变作为高水平扩增或缺失阈值,则共有58个区域检测到高水平扩增,主要是lp,2q,4q,5q,9p,10q, llq,13q,15p,15q, 19q,20p,21q,22q,而缺失则位于11q及Y染色体上。对于存在高水平扩增的DNA区域,通过查找胰腺表达数据库,筛查相关的候选基因,我们发现除Wnt-β-catenin信号通路相关的几个基因位点出现扩增外,还发现Notch 2所在染色体1p12区域易检测到高水平的扩增,其次为OR4F4等所在的15q26.3及OR4M1等所在的14q11区域易检测到高水平扩增。而在胰腺导管腺癌中易出现的KRAS,P53,P16及DPC4基因及CDKN2A, SMAD4, DCC等抑癌基因未能在SPN中检测到,提示在SPN的发生及发展中出现的分子遗传学改变与胰腺导管腺癌不同。
SPN是一种具有低度恶性潜能的胰腺实体性肿瘤,目前尚无判断其预后的形态学金标准,我们对作aCGH检测的10例SPN标本,以①肿瘤>5CM；②出现肿瘤性坏死；③侵犯胰腺周围软组织；④脉管/神经周围侵犯；⑤核分裂像增多或增殖指数增高作为具有侵袭性特征的组织学指标,我们发现具有较多侵袭性特征组织学指标的SPN样本易检测到DNA拷贝数改变,且出现高水平扩增者要多于无侵袭性组织学特征的SPN样本。
在aCGH初步筛选的基础上,为进一步探讨Notch信号通路在SPN发生发展中的作用,我们选择冷冻新鲜的SPN, PDAC, PET及正常胰腺组织样本各10例,用RT-PCR法检测了Notch信号通路的主要成分Notch1, Notch2, Notch4及Jagged-1 mRNA在这些样本中的表达,并选择SPN, PDAC, PET及正常胰腺组织各20例常规石蜡存档标本,用免疫组化法检测了Notch 1, Notch3, Jagged-1及CyclinD1蛋白在这些样本中的表达情况,我们发现Notch2 mRNA在SPN中表达明显上调,Notch1, Jagged-1 mRNA在SPN中表达亦上调,Notch4 mRNA的表达未见明显改变。Notch1, Jagged-1及CyclinD1蛋白在SPN中表达明显上调,而Notch3未见明显改变,提示Notch信号通路与SPN的发生有密切的关系,活化的Notch信号通路有助于维持SPN部分肿瘤细胞处于未分化的多潜能干细胞状态。Notch信号通路可能通过CyclinD1与Wnt-β-catenin信号通路协同作用,参与SPN的发生发展。免疫组化法检测Notch1及Jagged-1在SPN中呈强阳性,而在胰腺神经内分泌肿瘤中呈弱阳性或不表达,有助于我们运用此方法在日常病理诊断中将胰腺神经内分泌肿瘤与胰腺实性假乳头状肿瘤鉴别开来。
根据我们的实验结果,可以得出以下结论：
1.胰腺SPN可能起源于胰腺多潜能干细胞，并向胰腺外分泌，内分泌及神经嵴多向分化,其不同的免疫组化标记表达可能是多潜能干细胞沿不同方向或不同阶段分化的结果。
2.aCGH检测结果提示SPN除存在Wnt-β-catenin信号通路异常活化外,可能与Notch信号通路的活化有关,其发生发展中出现的分子遗传学改变与胰腺导管腺癌不同,组织学上具有较多的侵袭性特征者,aCGH易检测出高水平扩增。
3.Notch信号通路中的主要成分Notch1, Notch2及Jagged-1在SPN中呈高表达,Notch3, Notch4弱表达或不表达,提示Notch信号通路与SPN的发生发展有密切关系。免疫组化法检测Notch1, Jagged-1有助于我们在日常病理诊断中将胰腺实性假乳头状肿瘤与胰腺神经内分泌肿瘤鉴别开来。
Solid-pseudopapillary neoplasm(SPN) is a rare and low-grade malignant neoplasm.They occur predominanlly in young women,accounting for 1%-2% of all pancreatic neoplasm.They used to be misdiagnosed as no-functional islet cell tumor,cystic adenoma and papillary acinic cell carcinoma of the pancreas in the past. But their prognosis is much better than ductal adenocarcinoma and endocrine neoplasm of the pancreas. So, it has great signicance to understand the histogenesis and differential diagnosis of solid-pseudopapillary neoplasm of the pancreas.
The reported cases of SPN have been increased greatly duo to the realization of their morphology and wide use of new immunohistochemical markers recently. But the origin and pathogenesis of SPN are not clear. There are several speculations in the histogenesis of SPN such as ductal epithelial, acinic cell, endocrine cell, embryonic neural crest or pluri potent indifferent stem cell origin. The exploration of the histogenesis of SPN is a hotpoint of study of pancreatic neoplasms.
In 2001,Tanaka et al reported that almost all solid-pseudopapillary neoplasms harbor somatic point mutation in exon 3 of CTNNB1, the gene encodingβ-catenin protein that escapes intracytoplasmic phosphorylation and subsequent degradation and that therefore binds to the T-cell transcription factor(Tcf)/Lymphoid enhancer-binding factor(Lef).Theβ-catenin-Tcf/Lef complex is then abnormally translocated to the nucleus and activates the transcription of several oncogenic genes including MYC and Cyclin D1.This leads to the activation of the Wnt/β-catenin signaling pathways. The loss of membrane expression of (3-catenin on immunohistochemistry and neuclear expression ofβ-catenin of tumor cells may be helpful in establishing the diagnosis of a solid-pseudopapillary neoplasm of the pancreas. Aside from activation of the Wnt-β-catenin pathway, little is known about other molecular pathways, oncogenes or tumor suppressor genes involved in the pathogenesis of SPN.
DNA copy number variations (CNVs) play an important role in the pathogenesis and development of human genetic diseases and tumors. In recent years, array-based comparative genomic hybridization (aCGH) has been used to analyze the CNVs of tumor samples at a genomic scale and at progressively higher resolutions. Cy3-dCTP and Cy5-dCTP fluorescent dyes were used for labeling of the tumor DNA and reference DNA respectively and were mixed and co-hybridizaed with a high-resolution DNA microarray. Copy number aberrations of large or small genomic regions can be tested between tumor samples and reference DNA. Genetic changes including amplification or loss of genomic DNA of tumors or genetic diseases can be detected at a whole chromosome level. The current concept is that frequently occurring regions of DNA amplification commonly harbor oncogenes, whereas regions of recurrent deletion harbor tumor suppressor genes. This approach may aid in screen for novel candidate oncogenes or tumor suppressor genes involved in the pathogenesis of tumors. Analysing SPN samples using aCGH to identify different types of genomic aberrations may help us to understand the pathogenesis and development of SPN.
From January 2003 to June 2010, there were 48 cases of SPN which were resected or biopsied and were pathologically confirmed in the First Affiliated Hospital of Zhejiang University. To explore the histogenesis of SPN, we selected 20 cases of SPN which had detailed history, follow-up information and were big enough for histological, immunohistochemical and ultrastructural analysis.15 cases of ductal adenocarcinoma and 15 cases of neuroendocrine tumor of pancreas were also selected as reference in immunohistochemical study. E-cadherin andβ-catenin were selected as specific SPN markers. CKpan was selected as pancreatic duct cell marker. Alpha-1-antitrypsin (α-1-AT) was selected as acinic cell marker. S-100 protein and Melan A were selected as neural crest and melanocytic markers. Chromogranin A, Synaptophysin, CD56 and Secretagogin were selected as neuroendocrine markers.91% of our cases was female patients and average age was 33.4 years. The size of our SPN cases ranged from 1.2'to 14 cm. The cut section reveals brown to yellow solid areas with haemorrage and cystic degeneration. Histologically, SPN are composed of poorly cohesive monomorphic cells forming solid and pseudopapillary structures.By electron microscope, the neoplastic cells contain abundant cytoplasm, which is rich in mitochondria, abundant rough endoplasmic reticulum and zymogen-like granules of variable size. Neurosecretory-like granules have not been found. Immunohistochemically, all SPN express nuclear/cytoplasmicβ-catenin and loss of membrane expression of E-cadherin.80% (16/20) cases of SPN neoplastic cells are positive for CKpan, but staining is of a weak intensity compared with normal pancreatic epithelium.95%(19/20) cases of SPN are positive forα-1-antitrypsin but exhibiting sparsely granular staining in the cytoplasm with a focal localization. Immunoreactivity for CD56 is 100% positive and Synaptophysin is positive in 55%cases of SPN. But the specific neuroendocrine marker Chromogranin A is positive in 5%(1/20) cases of SPN and the new neuroendocrine marker Secretagogin is negative in all cases of SPN studied. S100 protein expressed in 5%(1/20) cases of SPN and Melan A are expressed in 10%(2/20) cases of SPN. Our morphological and immunohistochemical results support the hypothesis that SPN may derived from a pluripotent indifferent stem cell capable of exocrine, endocrine and neural crest differentiation. The different expression profiles of tumor cells for immunohistochemical markers may be the results of the pluripotent indifferent stem cells differentiated at different direction or at different stage.
In order to investigate the molecular pathways, oncogenes, and tumor suppressor genes involved in the pathogenesis and development of SPN aside from activation of the Wnt-β-catenin pathway, we carried out high-resulation aCGH analysis in 10 cases of SPN. In total,112 regions of CNVs were identified in 10 SPN samples. The average number of alterations per tumor was 22.3, ranging from 12 to 53 regions per case. The frequent amplifications were detected on chromosome arms 1p,2q,4q,5q,8p,9p, 1Oq, 11q,13q,15p,15q,16p,19q,20p,21q,22q and Xp. The frequent losses were observed on chromosome arms 11q,17p,20p and chromosome Y. The threshold for genetic "gains" or "losses" was determined as log 2 ratio≧0.2 or≦-0.2 respectively. High-level amplifications or homozygous deletions were defined as a log 2 ratio≧0.75 or≦-0.75 respectively, which corresponds to a theoretical±3.5 fold of the threshold for low-level amplifications.58 regions of high-level amplifications were identified, most of which were located on 1p,2q,4q,5q,9p, 10q, 11q,13q,15p,15q,19q,20p,21q and 22q. Homozygous deletions were observed on 11q and chromosome Y. Regions of frequent high -level amplifications and homozygous deletions were screened for candidated genes using the pancreastic expression database. We found aberrant activation of the Wnt-p-catenin pathway in SPN led to increased expression of several genes associated with Wnt pathway. High -level amplifications can be detected on chromosome 1p12, where harbor Notch 2 gene,15q26.3, where contain OR4F4 and 14q11, where contain OR4M1 gene. Alterations in the KRAS, TP53, P16, DPC4 and CDKN2A, SMAD4, DCC genes, frequently found in ductal adenocarcinomas of pancreas, have not been identified in SPN. Our results suggested that the genetic alterations in the pathogenesis of SPN were different from those of ductal adenocarcinoma of the pancreas.
Solid-pseudopapillary neoplasms of the pancreas are low-grade malignant tumors, their biological behavior can not be stratified solely on the basis of histopathological criteria. Based on a literature search, we selected the following histopathological features as indicators of potentially aggressive tumor biology of our 10 cases of SPN performing aCGH analysis.(1) tumor size > 5cm.②tumor necrosis.③invasion into peripancreatic soft tissues.④lymphvascular/perineural invasion.⑤increased mitotic /proliferative rate. We found that SPN with aggressive histopathological features tend to have more DNA CNVs. High-level amplifications can be easily detected by aCGH in SPN samples with more aggressive histopathological features.
To further confirm the results obtained from our aCGH analysis and to explore the role of Notch signaling pathway in the pathogenesis and development of SPN, we selected 10 cases of frozen specimens of SPN, PDAC, PET and normal pancreatic tissues respectively for RT-PCR study. The expression of members of Notch pathway (Notch 1, Notch2, Notch4 and Jagged-1) mRNA were detected using RT-PCR. The expression of Notchl, Notch 3, Jagged-1 and CyclinDl proteins were detected in 20 cases of SPN, PDAC, PET and normal pancreatic tissues respectively using immunohistochemistry. RT-PCR revealed over-expression of Notch 2 and Jagged-1 mRNA relative to the normal pancreatic tissues. Notch 1 mRNA over-expression was confirmed in SPN, although this was also observed in PDAC.Notch 4 expression was up-regulated and this was not observed in PDAC and PET. Immunohistochemistry revealed strong positive staining of Notch 1, Jagged-1 and Cyclin D1 proteins in SPN.Notch3 protein showed weak positive staining in SPN, PET, PDAC and normal pancreatic tissues. Our findings suggested that Notch signaling pathway was involved in the pathogenesis of SPN. Activated Notch in SPN may contribute to the maintenance of undifferentiated pluripotent stem cells. Notch signaling pathway and Wnt-β-catenin pathway may be activated in SPN through CyclinDl.Strong immunostaining of Notchl and Jagged-1 in SPN may be helpful to distinguish SPN from PET in our routine pathological diagnosis.
Based on the results of our experiments, we concluded:
1. SPN may be derived from a pluripotent indifferent stem cell capable of exocrine, endocrine and neural crest differentiation. The different expression profiles of tumor cells for immunohistochemical markers may be the results of the pluripotent indifferent stem cells differentiated at different direction or at different stage.
2. Aside from activation of the Wnt-β-catenin pathway, aCGH analysis results suggested that Notch pathway was also activated in SPN. The genetic alterations in the pathogenesis of SPN were different from those of ductal adenocarcinomas of the pancreas. High-level amplifications can be easily detected in SPNs with more aggressive histopathological features.
3. Members of Notch pathway (Notch 2, Notch 1, and Jagged-1) were up-regulated in SPN. Notch 3 and Notch 4 were weakly expressed in SPN. Our results suggested that Notch signaling pathway was involved in the pathogenesis of SPN. The differential immunstaining of Notch 1 and Jagged-1 proteins may be helpful to distinguish SPN from PET in our routine pathological diagnosis.
引文
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