Detection of Somatic Mutations in Gastroenteropancreatic

ANTICANCER RESEARCH 37 : 705-712 (2017) doi:10.21873/anticanres.11367 Detection of Somatic Mutations in Gastroenteropancreatic Neuroendocrine Tumors Using Targeted Deep Sequencing SAMUEL BACKMAN 1, OLOV NORLÉN 1, BARBRO ERIKSSON 2, BRITT SKOGSEID 2, PETER STÅLBERG 1 and JOAKIM CRONA 1 Department of 1Surgical and 2Medical Sciences, Uppsala University, Uppsala, Sweden Abstract. Mutations affecting the mechanistic target of in NETs and response to everolimus should be investigated rapamycin (MTOR) signalling pathway are frequent in by future studies. human cancer and have been identified in up to 15% of pancreatic neuroendocrine tumours (NETs). Grade A Aberrant activation of the mechanistic target of rapamycin evidence supports the efficacy of MTOR inhibition with (MTOR) pathway has been identified in multiple different everolimus in pancreatic NETs. Although a significant cancer types (1) and may occur as a result from both loss of proportion of patients experience disease stabilization, only function [phosphatase and tensin homolog ( PTEN ), tuberous a minority will show objective tumour responses. It has been sclerosis 1 ( TSC1 ) and TSC2 and gain of function proposed that genomic mutations resulting in activation of [phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic MTOR signalling could be used to predict sensitivity to subunit alpha ( PIK3CA ), MTOR mutations (2, 3). Inhibitors everolimus. Patients and Methods: Patients with NETs that of the MTOR signalling complex (rapalogues), such as underwent treatment with everolimus at our Institution were everolimus, have shown clinical benefit in a variety of cancer identified and those with available tumour tissue were types, including renal and breast carcinomas, as well as selected for further analysis. Targeted next-generation neuroendocrine tumours (NETs) of pancreatic origin (4-7). sequencing (NGS) was used to re-sequence 22 genes that Gastroenteropancreatic NETs are relatively rare tumours, were selected on the basis of documented involvement in the and the majority of patients show advanced disease stage MTOR signalling pathway or in the tumourigenesis of upon diagnosis (8, 9). The treatment arsenal for metastatic gastroenterpancreatic NETs. Radiological responses were disease includes somatostatin analogues, alkylating documented using Response Evaluation Criteria in Solid chemotherapy and signalling pathway inhibitors (4, 10-12). Tumours. Results: Six patients were identified, one had a Grade A evidence supports the antitumor effect of the partial response and four had stable disease. Sequencing of rapalogue everolimus in pancreatic NETs (4, 13). Although tumour tissue resulted in a median sequence depth of 667.1 a majority of patients with pancreatic NET in the RADIANT (range=404-1301) with 1-fold coverage of 95.9-96.5% and 3 trial experienced disease stabilization, only 5% had a 10-fold coverage of 87.6-92.2%. A total of 494 genetic significant tumour reduction (4). Everolimus is also variants were discovered, four of which were identified as approved by the US Food and Drug Administration for pathogenic. All pathogenic variants were validated using treatment of non-functional lung and gastrointestinal NETs Sanger sequencing and were found exclusively in menin 1 following results from the RADIANT 4 trial (14). In order (MEN1) and death domain associated protein (DAXX) genes. to avoid administration of a less effective treatment No mutations in the MTOR pathway-related genes were associated with significant morbidity, efficient biomarkers observed. Conclusion: Targeted NGS is a feasible method capable of predicting NET sensitivity to everolimus are with high diagnostic yield for genetic characterization of being sought. Recent studies in vitro have shown that pancreatic NETs. A potential association between mutations reduced phosphorylation of the MTOR complex can predict resistance to everolimus in bronchial NETs (15). Furthermore, high-throughput sequencing studies of epithelial cancer have revealed that somatic mutations in Correspondence to: Joakim Crona, MD, Ph.D, Department of genes associated with MTOR signalling can confer Surgical Sciences, Akademiska sjukhuset ing 70, FOA2, 75185, Uppsala, Sweden. Tel: +46 186110000, Fax: +46 186114911, e-mail: sensitivity to everolimus therapy (16-18). Iyer et al. [email protected] sequenced the exomes of bladder carcinomas and identified TSC1 mutations exclusively in responders to everolimus Key Words: MTOR, biomarker, PNET, next-generation sequencing. therapy (16). Wagle et al. utilized a different approach and 705 ANTICANCER RESEARCH 37 : 705-712 (2017) sequenced tumours from exceptional rapalogue responders, and 4), endothelial PAS domain protein 1 ( EPAS1 ), von Hippel- revealing an activating MTOR mutation in a patient with Lindau disease tumour suppressor ( VHL ), PIK3CA (codons 545, metastatic urothelial carcinoma (17). 1047), catenin beta 1 ( CTNNB1 ; exons 3 and 5), KIT proto-oncogene receptor tyrosine kinase ( KIT ; exons 9, 11, 13, 17), rapamycin- While the majority of pancreatic NETs have mutations in insensitive companion of MTOR ( RICTOR ), fibroblast growth factor chromatin-modulating genes [menin 1 ( MEN1 ) and death receptor 4 ( FGFR4 ; codon 388), DAXX , serine/threonine-protein domain-associated protein ( DAXX ) and chromatin remodeler kinase B-raf ( BRAF ; codon 600), epidermal growth factor receptor (ATRX ) genes], about 15% also harbour mutations in genes (EGFR ; codons 18-21), TSC1, PTEN, MEN1, GTPase HRAS involved in MTOR signalling (19-22). This was different (HRAS ; (exons 2-4), GTPase KRAS ( KRAS ; exons 2-4), RAC-alpha from SI NETs, in which the only significantly mutated gene serine/threonine-protein kinase ( AKT1 ; codon 17), transcriptional was cyclin-dependent kinase inhibitor 1B ( CDKN1B ), repressor protein YY1 ( YY1 ; codon 372), TSC2 , regulatory- associated protein of MTOR complex 1 ( RPTOR ) and cellular involved in cell-cycle regulation (23). There was no tumour antigen p53 ( TP53 ). Full details are presented in Table II. In significant association with mutations in genes related to the order to be able to detect variants causing alternative splicing in MTOR pathway in small intestinal (SI) NETs (23-25). We tumour-suppressor genes, coordinates were extended with a padding analyzed tumour specimens from patients with NET treated of 10 base pairs at intron–exon boundaries. Coordinates were with everolimus for mutations in disease-specific and MTOR obtained from the human reference sequence HG19 and the pathway genes. cumulative target size was 52040 base pairs. The final TruSeq Custom Amplicon design constituted 672 amplicons having a median Patients and Methods size of 175 bases. The in silico amplicon coverage was >98% with a total gap distance of 827 bases located in regions with homologous Patients and tissues. This was a retrospective study of patients treated sequences: RPTOR exon 6, MEN1 exon 2 and TP53 exon 6. at the Department of Endocrine Surgery that included biomaterial from Uppsala Biobank, Endocrine tumour collection (Ethical Library preparation and MiSEQ sequencing . Library preparation approval 00-128/3.15.2000). The study was approved by the local and sequencing was performed at the university core facility Ethical Committee (dnr no. 2011/375 and 2012/160). Written- (http://molmed.medsci.uu.se/SNP+SEQ+Technology+Platform/) as informed consent was obtained from the individual patients. All detailed in the MiSEQ System user guide for Reagent Kit v2. patients were above 18 years of age at the time of inclusion. Frozen Targeted enrichment and library preparation were performed from tumour tissue from patients with histopathologically confirmed NET 250 ng of double-stranded DNA (Illumina Inc.) according to the who had been treated with everolimus (n=6) were identified. manufacturer’s instructions. Briefly, upstream and downstream Cryosections were obtained and analysed with haematoxylin and oligonucleotides were hybridized to genomic DNA followed by an eosin stain to confirm a tumour cell content of >50%. Characteristics extension-ligation process that connected the hybridized upstream of the included patients are outlined in Table I. and downstream oligonucleotides using a DNA ligase. Extension- ligation fragments were amplified by polymerase chain reaction Response criteria. Responses to therapy were evaluated using (PCR) and connected to index adaptor sequences to allow for Response Evaluation Criteria In Solid Tumours (RECIST) 1.1 sample multiplexing using the TruSeq Custom Amplicon Index Kit Criteria. Briefly, the computed tomographic/magnetic resonance (Illumina Inc.). The PCR product was purified using AMPure XP images after therapy were compared to those at baseline and rated beads (Beckman Coulter Inc, Carlsbad, CA, USA). To confirm as: complete response, with a disappearance of all lesions; partial successful library preparation selected test samples were separated response if the longest diameter of index lesions decreased by and visualized on a 4% agarose gel. Each library sample underwent ≥30%; progressive disease if the longest diameter of target lesions quantity normalization and all six samples were pooled together increased by ≥20%; and stable disease if the sum of index lesions with 88 samples of other origin into a single suspension. The was between these parameters (26). generated paired end libraries were subjected to a single sequencing using an Illumina MiSEQ instrument (Illumina Inc.). Generated DNA extraction. Genomic DNA was extracted

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