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Prognostic Impact of Novel Molecular Subtypes of Small Intestinal Published OnlineFirst July 13, 2015; DOI: 10.1158/1078-0432.CCR-15-0373 Biology of Human Tumors Clinical Cancer Research Prognostic Impact of Novel Molecular Subtypes of Small Intestinal Neuroendocrine Tumor Anna Karpathakis1,2, Harpreet Dibra1, Chistodoulos Pipinikas1, Andrew Feber1, Tiffany Morris1, Joshua Francis3,4, Dahmane Oukrif1, Dalvinder Mandair1,2, Marinos Pericleous2, Mullan Mohmaduvesh2, Stefano Serra5, Olagunju Ogunbiyi2, Marco Novelli1, TuVinh Luong2, Sylvia L. Asa5, Matthew Kulke4, Christos Toumpanakis2, Tim Meyer1,2, Martyn Caplin2, Matthew Meyerson3,4, Stephan Beck1, and Christina Thirlwell1,2 Abstract Purpose: Small intestinal neuroendocrine tumors (SINET) are BeadChip (Illumina) array profiling (n ¼ 69), methylated DNA the commonest malignancy of the small intestine; however, immunoprecipitation sequencing (n ¼ 16), copy-number var- underlying pathogenic mechanisms remain poorly characterized. iance analysis (n ¼ 47), and Whole-Genome DASL (Illumina) Whole-genome and -exome sequencing has demonstrated that expression array profiling (n ¼ 43). SINETs are mutationally quiet, with the most frequent known Results: Based on molecular profiling, SINETs can be classified mutation in the cyclin-dependent kinase inhibitor 1B gene into three groups, which demonstrate significantly different pro- (CDKN1B) occurring in only 8% of tumors, suggesting that gression-free survival after resection of primary tumor (not alternative mechanisms may drive tumorigenesis. The aim of this reached at 10 years vs. 56 months vs. 21 months, P ¼ 0.04). study is to perform genome-wide molecular profiling of SINETs in Epimutations were found at a recurrence rate of up to 85%, and 21 order to identify pathogenic drivers based on molecular profiling. epigenetically dysregulated genes were identified, including This study represents the largest unbiased integrated genomic, CDX1 (86%), CELSR3 (84%), FBP1 (84%), and GIPR (74%). epigenomic, and transcriptomic analysis undertaken in this Conclusions: This is the first comprehensive integrated molec- tumor type. ular analysis of SINETs. We have demonstrated that these tumors Experimental Design: Here, we present data from integrated are highly epigenetically dysregulated. Furthermore, we have molecular analysis of SINETs (n ¼ 97), including whole-exome or identified novel molecular subtypes with significant impact on targeted CDKN1B sequencing (n ¼ 29), HumanMethylation450 progression-free survival. Clin Cancer Res; 1–9. Ó2015 AACR. Introduction inhibitor 1B) have been identified in approximately 8% of tumors in large-scale sequencing studies (3, 4). Mutations occurring in The incidence of small intestinal neuroendocrine tumors CDKN1B are loss-of-function truncating mutations that occur (SINET) is increasing (1) and accounts for 25% of all NETs. To throughout the gene. Mutational status does not appear to cor- date, the underlying pathogenic mechanisms have been poorly relate with expression of p27 (the protein product of CDKN1B) characterized, and due to relatively rarity, these tumors have not and has no detectable association with clinical characteristics or been subject to large-scale integrated genomic analyses, such as survival (4). Given the low frequency of mutations in this putative The Cancer Genome Atlas (TCGA) or International Cancer tumor suppressor gene, and the absence of other obvious path- Genome Consortium (ICGC) projects. ogenetic genome alterations, we hypothesized that alternative The most frequent genomic event identified in SINETs is loss mechanisms such as epigenetic dysregulation are likely to play a of heterozygosity (LOH) at chromosome 18, occurring in 60% role in these tumors (5). to 78% of tumors (2, 3); more recently, recurrent mutations Epigenetic alterations lead to changes in gene expression in the cell cycle regulator CDKN1B (cyclin-dependent kinase without modification of the underlying DNA sequence. DNA methylation and chromatin modifications are two of the mechanisms which regulate gene expression and are frequently 1University College London, London, United Kingdom. 2The Royal Free Hospital, London, United Kingdom. 3The Broad Institute of Harvard disrupted in cancer. It has long been recognized that hyper- and MIT, Cambridge, Massachusetts. 4Dana-Farber Cancer Institute, methylation at the promoter region of a gene can lead to 5 Harvard Medical School, Boston, Massachusetts. UHN Princess reduced gene expression (6). More recently, it has been dem- Margaret Cancer Centre, Toronto, Canada. onstrated that hypermethylation over the gene body leads Note: Supplementary data for this article are available at Clinical Cancer to increased gene expression, and demethylating the gene Research Online (http://clincancerres.aacrjournals.org/). body by application of 5-aza-2-deoxycytidine leads to down- Corresponding Author: Christina Thirlwell, University College London, 72 Hunt- regulation (7). ley St, London WC1E6BT, UK. Phone: 020-76796882; Fax: 020-76796884; Epigenetic dysregulation is known to play a key role in other E-mail: [email protected] NET development, including tumors of pancreatic and bronchial doi: 10.1158/1078-0432.CCR-15-0373 origin (8, 9). Integrated molecular profiling based on methylation Ó2015 American Association for Cancer Research. analysis has recently been shown to identify clinically distinct www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst July 13, 2015; DOI: 10.1158/1078-0432.CCR-15-0373 Karpathakis et al. RNA Paraffin kit). Where nucleic acids were extracted from for- Translational Relevance malin-fixed paraffin-embedded (FFPE) tissue blocks, 6 to 12 Â 6 To date, limited understanding of the underlying biology of mm serial sections were cut and macrodissected, ensuring that SINETs has hindered personalized management. The most >80% of the sample was histologically confirmed as tumor. Fresh frequent genetic mutation (CDKN1B, affecting 8% of tissue samples were frozen in liquid nitrogen within 30 minutes of tumors) has no immediate detectable impact on clinical resection. Serial hematoxylin and eosin–stained sections were phenotype or outcome. Due to their relative rarity, SINETs evaluated to ensure >80% purity of tumor and normal specimens. have not been included in any of the international collabo- rative cancer molecular profiling efforts. We have therefore DNA mutation analysis: whole-exome and targeted CDKN1B performed a Cancer Genome Atlas style–integrated analysis of sequencing a large cohort of SINETs in order to comprehensively charac- Exome sequencing was performed by Joshua Francis on 1 mg terize their molecular profile. We identified three molecular DNA as previously described (3). In brief, exonic region capture subtypes of SINET that are associated with significant differ- was performed utilizing the Agilent V2 capture probe set, and ence in progression-free survival after surgical resection. We sequencing of 76-bp paired end reads was performed utilizing also demonstrated that SINETs are highly epigenetically dys- Illumina HiSeq2000 instruments. A median of 9.15 Gb of unique regulated and characterized a panel of 21 genes that are sequence data was generated per sample, which was aligned to the epigenetically altered in 70% to 80% of cases. These findings target exome using the Burrows–Wheeler Aligner (BWA), resulting highlight the molecular diversity of SINETs and highlight in a median 140Â coverage of each base. Targeted sequencing was epigenetic mechanisms as potential drivers of tumorigenesis. performed by multiplex PCR reaction tiling of CDKN1B using 100 Novel molecular profiling could be implemented in the to 200 ng of DNA on the Fluidigm Access Array microfluidic clinical setting to facilitate personalized management and device. PCR products underwent Illumina sequencing on the improve prognosis. MiSeq instrument to a mean coverage of 800Â. DNA methylation analysis: Illumina HumanMethylation450 BeadChip subgroups of lung carcinomas, including a neuroendocrine-spe- DNA from SINET samples was analyzed on the HumMeth450 cific "epitype" (10). BeadChip (HM450). The integrity and viability of FFPE samples fi Here, we present the first comprehensive integrated genome- were rst determined using the Illumina FFPE QC Kit. Ligation of fi wide molecular analysis of a large cohort of SINETs. FFPE DNA (Qiagen REPLI-g FFPE Kit) and bisul te conversion of FF and FFPE DNA (Zymo EZ DNA Methylation kit) were per- fi Materials and Methods formed as described previously (12). Quality control of bisul te conversion was performed by quantitative PCR to confirm >98% Patient recruitment conversion. Data analysis was performed using the ChAMP pipe- All patients provided written informed consent for their tissue line (13). Samples where 5% of probes failed detection P value < to be analyzed in this study, which was approved by NHS Camden 0.01 filtering were excluded from further analysis. Normalization and Islington Community Research Ethics Committee (Ref: 09/ with BMIQ and batch correction with ComBat were performed H0722/27). A total of 97 tumors and 25 normal tissue samples according to pipeline defaults. The comparability of array-based were analyzed from 85 individuals. Inclusion criteria were histo- methylation analysis of FFPE and FF tissues has previously been pathologic diagnosis of SINET, and surgical resection of either demonstrated (12). primary tumor or liver metastasis. Exclusion criteria were biopsy CpG island methylator phenotype
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