Molecular characterization of metastatic pancreatic neuroendocrine tumors (PNETs) using whole-genome and transcriptome sequencing

Hui-Li Wong, Kevin C Yang, Yaoqing Shen, Eric Y Zhao, Jonathan M Loree, Hagen F Kennecke, Steve E Kalloger, Joanna M Karasinska, Howard J Lim, Andrew J Mungall, Xiaolan Feng, Janine M Davies, Kasmintan Schrader, Chen Zhou, Aly Karsan, Steven J M Jones, Janessa Laskin, Marco A Marra, David F Schaeffer, Sharon M Gorski, Daniel J Renouf, Hui-Li Wong, Kevin C Yang, Yaoqing Shen, Eric Y Zhao, Jonathan M Loree, Hagen F Kennecke, Steve E Kalloger, Joanna M Karasinska, Howard J Lim, Andrew J Mungall, Xiaolan Feng, Janine M Davies, Kasmintan Schrader, Chen Zhou, Aly Karsan, Steven J M Jones, Janessa Laskin, Marco A Marra, David F Schaeffer, Sharon M Gorski, Daniel J Renouf

Abstract

Pancreatic neuroendocrine tumors (PNETs) are a genomically and clinically heterogeneous group of pancreatic neoplasms often diagnosed with distant metastases. Recurrent somatic mutations, chromosomal aberrations, and gene expression signatures in PNETs have been described, but the clinical significance of these molecular changes is still poorly understood, and the clinical outcomes of PNET patients remain highly variable. To help identify the molecular factors that contribute to PNET progression and metastasis, and as part of an ongoing clinical trial at the BC Cancer Agency (clinicaltrials.gov ID: NCT02155621), the genomic and transcriptomic profiles of liver metastases from five patients (four PNETs and one neuroendocrine carcinoma) were analyzed. In four of the five cases, we identified biallelic loss of MEN1 and DAXX as well as recurrent regions with loss of heterozygosity. Several novel findings were observed, including focal amplification of MYCN concomitant with loss of APC and TP53 in one sample with wild-type MEN1 and DAXX Transcriptome analyses revealed up-regulation of MYCN target genes in this sample, confirming a MYCN-driven gene expression signature. We also identified a germline NTHL1 fusion event in one sample that resulted in a striking C>T mutation signature profile not previously reported in PNETs. These varying molecular alterations suggest different cellular pathways may contribute to PNET progression, consistent with the heterogeneous clinical nature of this disease. Furthermore, genomic profiles appeared to correlate well with treatment response, lending support to the role of prospective genotyping efforts to guide therapy in PNETs.

Keywords: neoplasm of the gastrointestinal tract; neuroendocrine neoplasm.

© 2018 Wong et al.; Published by Cold Spring Harbor Laboratory Press.

Figures

Figure 1.
Figure 1.
Clinical evolution and treatment of five patients with metastatic pancreatic neuroendocrine tumors and carcinomas enrolled in the Personalized Oncogenomics program. Cap/Tem, capecitabine with temozolomide; Cis/Etop, cisplatin with etoposide; Carbo/Iri, carboplatin with irinotecan.
Figure 2.
Figure 2.
Genome-wide copy-number architectures across the five cases. (A) Chromosomal regions with loss of heterozygosity events are depicted in green. The zygosity states at these regions are not discriminated. (B) Ploidy-corrected copy-number changes in protein-coding regions are depicted in red (copy gain) or blue (copy loss). The magnitude of copy-number gains is capped at +4.
Figure 3.
Figure 3.
Key molecular alterations and predicted upstream regulators across the five cases. (A) An OncoPrint depicting alterations in genes that have either been implicated in PNETs or are targets of conventional therapeutic agents used to treat PNETs. All alterations described here are somatic, aside from Case 3, which had germline one copy loss of TSC2. “Set A” contains genes recurrently mutated in sporadic PNETs (Jiao et al. 2011). “Set B” contains genes in which germline mutations were recently reported in PNET patients (Scarpa et al. 2017). “Others” contains genes of interest in this study. “Drugs” contains genes with protein products that are the molecular targets (color-coded) of the indicated therapeutic agents used to treat PNETs. Nonsynonymous mutations (black bar), copy-number aberrations (red/blue shade), and up- or down-regulated expressions (red/blue edge) of these genes are shown. All genes have gray background by default to facilitate visualization. Shallow and deep deletions refer to one- or two-copy losses, respectively. Up-regulated genes are defined as those expressed at levels >90% of TCGA tumor compendium, and down-regulated genes as those expressed at levels <10% of TCGA tumor compendium. (B) Using the Upstream Regulator Analysis tool from Ingenuity Pathway Analysis software, the activation states of prospective upstream regulators in each sample were predicted based on differentially regulated genes. Only upstream regulators with activation score of >3 or <−3 were selected. Color and intensity indicate the predicted activation state and effect size, respectively. The overlap p-value of each prediction is also indicated. *, p < 0.05; **, p < 0.01; ***, p< 0.001; ****, p < 0.0001.

References

    1. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. 2010. A method and server for predicting damaging missense mutations. Nat Methods 7: 248–249.
    1. Alexandrov LB, Nik-Zainal S, Wedge DC, Campbell PJ, Stratton MR. 2013. Deciphering signatures of mutational processes operative in human cancer. Cell Rep 3: 246–259.
    1. Bergsland EK, Roy R, Stephens P, Ross JS, Bailey M, Olshen A. 2016. Genomic profiling to distinguish poorly differentiated neuroendocrine carcinomas arising in different sites. J Clin Oncol doi: 10.1200/JCO.2016.34.15_suppl.4020.
    1. Boon K, Caron HN, van Asperen R, Valentijn L, Hermus M-C, van Sluis P, Roobeek I, Weis I, Voûte PA, Schwab M, Versteeg R. 2001. N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis. EMBO J 20: 1383–1393.
    1. Chen L, Iraci N, Gherardi S, Gamble LD, Wood KM, Perini G, Lunec J, Tweddle DA. 2010. p53 is a direct transcriptional target of MYCN in neuroblastoma. Cancer Res 70: 1377–1388.
    1. Coriat R, Walter T, Terris B, Couvelard A, Ruszniewski P. 2016. Gastroenteropancreatic well-differentiated grade 3 neuroendocrine tumors: review and position statement. Oncologist 21: 1191–1199.
    1. Cros J, Hentic O, Rebours V, Zappa M, Gille N, Theou-Anton N, Vernerey D, Maire F, Levy P, Bedossa P, et al. 2016. MGMT expression predicts response to temozolomide in pancreatic neuroendocrine tumors. Endocr Relat Cancer 23: 625–633.
    1. Drost J, van Boxtel R, Blokzijl F, Mizutani T, Sasaki N, Sasselli V, de Ligt J, Behjati S, Grolleman JE, van Wezel T, et al. 2017. Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer. Science 358: 234–238.
    1. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M, et al. 2009. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 45: 228–247.
    1. Fielitz K, Althoff K, De Preter K, Nonnekens J, Ohli J, Elges S, Hartmann W, Kloppel G, Knosel T, Schulte M, et al. 2016. Characterization of pancreatic glucagon-producing tumors and pituitary gland tumors in transgenic mice overexpressing MYCN in hGFAP-positive cells. Oncotarget 7: 74415–74426.
    1. Forbes SA, Beare D, Gunasekaran P, Leung K, Bindal N, Boutselakis H, Ding M, Bamford S, Cole C, Ward S, et al. 2015. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Res 43: D805–D811.
    1. Gamble LD, Kees UR, Tweddle DA, Lunec J. 2012. MYCN sensitizes neuroblastoma to the MDM2-p53 antagonists Nutlin-3 and MI-63. Oncogene 31: 752–763.
    1. Gu Z, Eils R, Schlesner M. 2016. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32: 2847–2849.
    1. Ha G, Roth A, Lai D, Bashashati A, Ding J, Goya R, Giuliany R, Rosner J, Oloumi A, Shumansky K, et al. 2012. Integrative analysis of genome-wide loss of heterozygosity and monoallelic expression at nucleotide resolution reveals disrupted pathways in triple-negative breast cancer. Genome Res 22: 1995–2007.
    1. Halfdanarson TR, Rabe KG, Rubin J, Petersen GM. 2008. Pancreatic neuroendocrine tumors (PNETs): incidence, prognosis and recent trend toward improved survival. Ann Oncol 19: 1727–1733.
    1. Hegi ME, Diserens A-C, Gorlia T, Hamou M-F, de Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, et al. 2005. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352: 997–1003.
    1. Hicks RJ, Kwekkeboom DJ, Krenning E, Bodei L, Grozinsky-Glasberg S, Arnold R, Borbath I, Cwikla J, Toumpanakis C, Kaltsas G, et al. 2017. ENETS consensus guidelines for the standards of care in neuroendocrine neoplasia: peptide receptor radionuclide therapy with radiolabelled somatostatin analogues. Neuroendocrinology 105: 295–309.
    1. Huang M, Weiss WA. 2013. Neuroblastoma and MYCN. Cold Spring Harb Perspect Med 3: a014415.
    1. Jensen RT, Berna MJ, Bingham DB, Norton JA. 2008. Inherited pancreatic endocrine tumor syndromes: advances in molecular pathogenesis, diagnosis, management, and controversies. Cancer 113: 1807–1843.
    1. Jiao Y, Shi C, Edil BH, de Wilde RF, Klimstra DS, Maitra A, Schulick RD, Tang LH, Wolfgang CL, Choti MA, et al. 2011. DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors. Science 331: 1199–1203.
    1. Klimstra DS, Modlin IR, Coppola D, Lloyd RV, Suster S. 2010. The pathologic classification of neuroendocrine tumors: a review of nomenclature, grading, and staging systems. Pancreas 39: 707–712.
    1. Krämer A, Green J, Pollard J, Tugendreich S. 2014. Causal analysis approaches in Ingenuity Pathway Analysis. Bioinformatics 30: 523–530.
    1. Kulke MH, Hornick JL, Frauenhoffer C, Hooshmand S, Ryan DP, Enzinger PC, Meyerhardt JA, Clark JW, Stuart K, Fuchs CS, Redston MS. 2009. O6-methylguanine DNA methyltransferase deficiency and response to temozolomide-based therapy in patients with neuroendocrine tumors. Clin Cancer Res 15: 338–345.
    1. Li H, Durbin R. 2010. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26: 589–595.
    1. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25: 2078–2079.
    1. Luo W, Friedman MS, Shedden K, Hankenson KD, Woolf PJ. 2009. GAGE: generally applicable gene set enrichment for pathway analysis. BMC Bioinformatics 10: 161.
    1. Marinoni I, Kurrer AS, Vassella E, Dettmer M, Rudolph T, Banz V, Hunger F, Pasquinelli S, Speel EJ, Perren A. 2014. Loss of DAXX and ATRX are associated with chromosome instability and reduced survival of patients with pancreatic neuroendocrine tumors. Gastroenterology 146: 453–460.
    1. Missiaglia E, Dalai I, Barbi S, Beghelli S, Falconi M, della Peruta M, Piemonti L, Capurso G, Di Florio A, dell Fave G, et al. 2010. Pancreatic endocrine tumors: expression profiling evidences a role for AKT-mTOR pathway. J Clin Oncol 28: 245–255.
    1. Rausch T, Zichner T, Schlattl A, Stütz AM, Benes V, Korbel JO. 2012. DELLY: structural variant discovery by integrated paired-end and split-read analysis. Bioinformatics 28: i333–i339.
    1. Rivera B, Castellsague E, Bah I, van Kempen LC, Foulkes WD. 2015. Biallelic NTHL1 mutations in a woman with multiple primary tumors. N Engl J Med 373: 1985–1986.
    1. Robertson G, Schein J, Chiu R, Corbett R, Field M, Jackman SD, Mungall K, Lee S, Okada HM, Qian JQ, et al. 2010. De novo assembly and analysis of RNA-seq data. Nat Methods 7: 909–912.
    1. Sangoi AR, Ohgami RS, Pai RK, Beck AH, McKenney JK, Pai RK. 2011. PAX8 expression reliably distinguishes pancreatic well-differentiated neuroendocrine tumors from ileal and pulmonary well-differentiated neuroendocrine tumors and pancreatic acinar cell carcinoma. Mod Pathol 24: 412–424.
    1. Santos R, Ursu O, Gaulton A, Bento AP, Donadi RS, Bologa CG, Karlsson A, Al-Lazikani B, Hersey A, Oprea TI, Overington JP. 2017. A comprehensive map of molecular drug targets. Nat Rev Drug Discov 16: 19–34.
    1. Saunders CT, Wong WS, Swamy S, Becq J, Murray LJ, Cheetham RK. 2012. Strelka: accurate somatic small-variant calling from sequenced tumor-normal sample pairs. Bioinformatics 28: 1811–1817.
    1. Scarpa A, Chang DK, Nones K, Corbo V, Patch AM, Bailey P, Lawlor RT, Johns AL, Miller DK, Mafficini A, et al. 2017. Whole-genome landscape of pancreatic neuroendocrine tumours. Nature 543: 65–71.
    1. Sheffield BS, Tinker AV, Shen Y, Hwang H, Li-Chang HH, Pleasance E, Ch'ng C, Lum A, Lorette J, McConnell YJ, et al. 2015. Personalized oncogenomics: clinical experience with malignant peritoneal mesothelioma using whole genome sequencing. PLoS One 10: e0119689.
    1. Singhi AD, Liu T, Roncaioli JL, Cao D, Zeh HJ, Zureikat AH, Tsung A, Marsh JW, Lee KK, Hogg ME, et al. 2017. Alternative lengthening of telomeres and loss of DAXX/ATRX expression predicts metastatic disease and poor survival in patients with pancreatic neuroendocrine tumors. Clin Cancer Res 23: 600–609.
    1. Skidmore ZL, Wagner AH, Lesurf R, Campbell KM, Kunisaki J, Griffith OL, Griffith M. 2016. GenVisR: Genomic Visualizations in R. Bioinformatics 32: 3012–3014.
    1. Tang LH, Basturk O, Sue JJ, Klimstra DS. 2016. A practical approach to the classification of WHO grade 3 (G3) well-differentiated neuroendocrine tumor (WD-NET) and poorly differentiated neuroendocrine carcinoma (PD-NEC) of the pancreas. Am J Surg Pathol 40: 1192–1202.
    1. Tolcher AW, Papadopoulos KP, Patnaik A, Rasco DW, Martinez D, Wood DL, Fielman B, Sharma M, Janisch LA, Brown BD, et al. 2015. Safety and activity of DCR-MYC, a first-in-class dicer-substrate small interfering RNA (DsiRNA) targeting MYC, in a phase I study in patients with advanced solid tumors. J Clin Oncol doi: 10.1200/jco.2015.33.15_suppl.11006.
    1. Weren RD, Ligtenberg MJ, Kets CM, de Voer RM, Verwiel ET, Spruijt L, van Zelst-Stams WA, Jongmans MC, Gilissen C, Hehir-Kwa JY, et al. 2015. A germline homozygous mutation in the base-excision repair gene NTHL1 causes adenomatous polyposis and colorectal cancer. Nat Genet 47: 668–671.
    1. Yachida S, Vakiani E, White CM, Zhong Y, Saunders T, Morgan R, de Wilde RF, Maitra A, Hicks J, Demarzo AM, et al. 2012. Small cell and large cell neuroendocrine carcinomas of the pancreas are genetically similar and distinct from well-differentiated pancreatic neuroendocrine tumors. Am J Surg Pathol 36: 173–184.
    1. Yamaguchi H, Kawazu M, Yasuda T, Soda M, Ueno T, Kojima S, Yashiro M, Yoshino I, Ishikawa Y, Sai E, Mano H. 2015. Transforming somatic mutations of mammalian target of rapamycin kinase in human cancer. Cancer Sci 106: 1687–1692.
    1. Yang HW, Kutok JL, Lee NH, Piao HY, Fletcher CDM, Kanki JP, Look AT. 2004. Targeted expression of human MYCN selectively causes pancreatic neuroendocrine tumors in transgenic zebrafish. Cancer Res 64: 7256–7262.
    1. Yao JC, Shah MH, Ito T, Bohas CL, Wolin EM, Van Cutsem E, Hobday TJ, Okusaka T, Capdevila J, de Vries EG, et al. 2011. Everolimus for advanced pancreatic neuroendocrine tumors. N Engl J Med 364: 514–523.

Source: PubMed

Szponzorok és közreműködők

Egészségi állapot

Kábítószer-beavatkozások

3
Iratkozz fel