外显子组测序胃泌瘤素

NIH Public AccessAuthor ManuscriptScience. Author manuscript; available in PMC 2011 July 27.Published in final edited form as: Science. 2011 March 4; 331(6021): 1199–1203. doi:10.1126/science.1200609.

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DAXX/ATRX, MEN1 and mTOR Pathway Genes are Frequently Altered in Pancreatic Neuroendocrine TumorsYuchen Jiao1,*, Chanjuan Shi2,*, Barish H. Edil3, Roeland F. de Wilde2, David S. Klimstra4, Anirban Maitra5, Richard D. Schulick3, Laura H. Tang4, Christopher L. Wolfgang3, Michael A. Choti3, Victor E. Velculescu1, Luis A. Diaz Jr.1,6, Bert Vogelstein1, Kenneth W. Kinzler1,+, Ralph H. Hruban5,+, and Nickolas Papadopoulos1,+ 1Ludwig Center for Cancer Genetics and Howard Hughes Medical Institutions, Johns Hopkins Kimmel Cancer Center, Baltimore, MD 212312Department

of Pathology, the Sol Goldman Pancreatic Cancer Research Center, the Johns Hopkins Medical Institutions, Baltimore, MD 212313Department

of Surgery, the Sol Goldman Pancreatic Cancer Research Center, the Johns Hopkins Medical Institutions, Baltimore, MD 212314Department

of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065

5Departments

of Pathology and Oncology, the Sol Goldman Pancreatic Cancer Research Center, the Johns Hopkins Medical Institutions, Baltimore, MD 212316Swim

Across America Laboratory at Johns Hopkins, Baltimore, MD 21231

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asymptomatic abdominal mass or symptoms of abdominal pain secondary to compression bya large tumor. Surgical resection is the treatment of choice, but many patients present withunresectable tumors or extensive metastatic disease, and medical therapies are relativelyineffective in these cases.

There is currently insufficient information about this tumor to either predict prognosis ofpatients diagnosed with PanNETs or to develop companion diagnostics and personalizedtreatments to improve disease management. Biallelic inactivation of the MEN1 gene, usuallythrough a mutation in one allele coupled with loss of the remaining wild-type allele, occursin 25-30% of PanNETs (5, 6). MEN1 is a tumor suppressor gene which, when mutated in thegermline, predisposes to multiple endocrine neoplasia type 1 syndrome. Chromosomal gainsand losses and expression analyses have revealed candidate loci for genes involved in thedevelopment of PanNETs, but these have not been substantiated by genetic or functionalanalyses (7-9).

To gain insights into the genetic basis of this tumor type, we determined the exomicsequence of ~18,000 protein-coding genes in a Discovery set of ten well-characterizedsporadic PanNETs. A clinically homogeneous set of tumors of high neoplastic cellularity isessential for the successful identification of genes and pathways involved in any tumor type.Thus, we excluded small cell and large cell neuroendocrine carcinomas and studied onlysamples that were not part of a familial syndrome associated with PanNETs (table S1) (1).We microdisected tumor samples to achieve a neoplastic cellularity of >80%. DNA from theenriched neoplastic samples and from matched non-neoplastic tissue from ten patients wasused to prepare fragment libraries suitable for massively parallel sequencing. The codingsequences were enriched by capture with the SureSelect Enrichment System and sequencedusing an Illumina GAIIx platform (10). The average coverage of each base in the targetedregions was 101-fold and 94.8 % of the bases were represented by at least 10 reads (tableS2).

We identified 157 somatic mutations in 149 genes among the ten tumors used in the

Discovery set. The mutations per tumor ranged from 8 to 23, with a mean of 16 (table S3).Of these mutations, 91 % were validated by Sanger sequencing. There were some obviousdifferences between the genetic landscapes of PanNETs and those of pancreatic ductaladenocarcinomas (PDAC, ref. 11). First, there were 60% fewer genes mutated per tumor inPanNETs than in PDACs. Second, the genes most commonly affected by mutation in

PDACs (KRAS, TGF-β pathway, CDKN2A, TP53) were rarely altered in PanNETs and viceversa (Table 1). Third, the spectrum of mutations in PDAC and PanNET were different, withC to T transitions more common in PDACs than in PanNETs, and C to G transversions morecommon in PanNETs than in PDACs (table S4). This suggests that mutations in PanNETsand PDAC arise through different mechanisms, perhaps due to exposure to differentenvironmental carcinogens or through the action of different DNA repair pathways.We next selected genes for further analysis that were well-documented components of apathway that was genetically altered in more than one tumor, because alterations in thesegenes are most likely to be clinically relevant. Four genes were mutated in at least twotumors in the Discovery set: MEN1 in five, DAXX in three, PTEN in two, and TSC2 in two.ATRX was mutated in only one sample in the Discovery set, but its product forms a

heterodimer with DAXX and therefore is part of the same pathway, so it was also evaluatedin the Validation set. Similarly, PIK3CA was included because its product is part of themTOR pathway that includes PTEN and TSC2 (12-14). The sequences of these genes werethen determined by Sanger sequencing in a Validation set consisting of 58 additionalPanNETs and their corresponding normal tissues (Fig. 1, A and B). In total, somatic

Science. Author manuscript; available in PMC 2011 July 27.