Analysis of Mutation and Loss of Heterozygosity by Whole-Exome Sequencing Yields Insights into Pseudomyxoma Peritonei
2018; Elsevier BV; Volume: 20; Issue: 5 Linguagem: Inglês
10.1016/j.jmoldx.2018.05.002
ISSN1943-7811
AutoresReuben J. Pengelly, Babatunde Rowaiye, Karen Pickard, Brendan Moran, Sanjeev Dayal, William Tapper, Alex H. Mirnezami, Tom Cecil, Faheez Mohamed, Norman J. Carr, Sarah Ennis,
Tópico(s)Hepatocellular Carcinoma Treatment and Prognosis
ResumoPseudomyxoma peritonei (PMP) is a clinical syndrome characterized by gross mucinous ascites originating from a disseminated intraperitoneal neoplasm. Although typically confined to the abdomen, mortality is high if untreated. Biomarkers, including genetic mutation profiles, may aid treatment selection and decision making. We applied whole-exome sequencing to five patients diagnosed with low-grade appendiceal mucinous neoplasms, using paired tumor and germline samples identify biomarkers. Multiple bioinformatic approaches were applied to these data to assess both somatic mutation profiles and loss of heterozygosity events. Mutation profiles of the tumors were consistent with deamination of methylcytosine being the prevailing mechanism. Pathogenic mutations were identified in both KRAS and GNAS in all samples, and further mutations in genes implicated in PMP, namely FGFR2, APC, SMAD2, and FAT4. No TP53 somatic mutations were identified, matching expectations for low-grade tumors. Four of five samples exhibited clonal loss of heterozygosity; these regions were further examined and found to contain genes harboring pathogenic somatic mutations in some samples. RNF43 was hereby implicated in the pathogenesis of PMP of appendiceal origin, having previously been found to increase sensitivity to Wnt signaling and to have involvement in similar mucinous tumors. In conclusion, we have investigated the mutation profile of PMP of appendiceal origin and provided the first report of RNF43 involvement in its progression. Pseudomyxoma peritonei (PMP) is a clinical syndrome characterized by gross mucinous ascites originating from a disseminated intraperitoneal neoplasm. Although typically confined to the abdomen, mortality is high if untreated. Biomarkers, including genetic mutation profiles, may aid treatment selection and decision making. We applied whole-exome sequencing to five patients diagnosed with low-grade appendiceal mucinous neoplasms, using paired tumor and germline samples identify biomarkers. Multiple bioinformatic approaches were applied to these data to assess both somatic mutation profiles and loss of heterozygosity events. Mutation profiles of the tumors were consistent with deamination of methylcytosine being the prevailing mechanism. Pathogenic mutations were identified in both KRAS and GNAS in all samples, and further mutations in genes implicated in PMP, namely FGFR2, APC, SMAD2, and FAT4. No TP53 somatic mutations were identified, matching expectations for low-grade tumors. Four of five samples exhibited clonal loss of heterozygosity; these regions were further examined and found to contain genes harboring pathogenic somatic mutations in some samples. RNF43 was hereby implicated in the pathogenesis of PMP of appendiceal origin, having previously been found to increase sensitivity to Wnt signaling and to have involvement in similar mucinous tumors. In conclusion, we have investigated the mutation profile of PMP of appendiceal origin and provided the first report of RNF43 involvement in its progression. CME Accreditation Statement: This activity ("JMD 2018 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("JMD 2018 CME Program in Molecular Diagnostics") for a maximum of 18.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("JMD 2018 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2018 CME Program in Molecular Diagnostics") for a maximum of 18.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Pseudomyxoma peritonei (PMP) is a clinical condition characterized by mucinous ascites that originate from a disseminated intraperitoneal mucinous neoplasm The pathophysiology incorporates mucinous ascites, peritoneal tumor implants, gross omental involvement, and (in women) ovarian metastases.1Carr N.J. Finch J. Ilesley I.C. Chandrakumaran K. Mohamed F. Mirnezami A. Cecil T. Moran B. 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Proposed classification of pseudomyxoma peritonei: influence of signet ring cells on survival.Am Surg. 2013; 79: 1171-1176Crossref PubMed Google Scholar If untreated, PMP progresses, sometimes slowly but relentlessly, within the abdomen, ultimately leading to death, often by intestinal obstruction. The combination of cytoreductive surgery with hyperthermic intraperitoneal chemotherapy has markedly improved outcomes, especially if complete cytoreduction can be achieved.5Chua T.C. Moran B.J. Sugarbaker P.H. Levine E.A. Glehen O. Gilly F.N. Baratti D. Deraco M. Elias D. Sardi A. Liauw W. Yan T.D. Barrios P. Gómez Portilla A. de Hingh I.H. Ceelen W.P. Pelz J.O. Piso P. González-Moreno S. Van Der Speeten K. Morris D.L. 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Pathology and prognosis in pseudomyxoma peritonei: a review of 274 cases.J Clin Pathol. 2012; 65: 919-923Crossref PubMed Scopus (127) Google Scholar, 8Smeenk R.M. van Velthuysen M.L. Verwaal V.J. Zoetmulder F.A. Appendiceal neoplasms and pseudomyxoma peritonei: a population based study.Eur J Surg Oncol. 2008; 34: 196-201Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 9Misdraji J. Yantiss R.K. Graeme-Cook F.M. Balis U.J. Young R.H. Appendiceal mucinous neoplasms: a clinicopathologic analysis of 107 cases.Am J Surg Pathol. 2003; 27: 1089-1103Crossref PubMed Scopus (408) Google Scholar which can be classified as low-grade appendiceal mucinous neoplasm (LAMN), high-grade appendiceal mucinous neoplasm, or mucinous adenocarcinoma.10Carr N.J. Cecil T.D. Mohamed F. Sobin L.H. Sugarbaker P.H. González-Moreno S. Taflampas P. Chapman S. Moran B.J. Peritoneal Surface Oncology Group InternationalA consensus for classification and pathologic reporting of pseudomyxoma peritonei and associated appendiceal neoplasia: the results of the Peritoneal Surface Oncology Group International (PSOGI) modified Delphi process.Am J Surg Pathol. 2016; 40: 14-26Crossref PubMed Scopus (375) Google Scholar Certain mucins are more likely to be overproduced in PMP, with MUC2, MUC5AC, and MUC5B being the most common.11Amini A. Masoumi-Moghaddam S. Ehteda A. Morris D.L. Secreted mucins in pseudomyxoma peritonei: pathophysiological significance and potential therapeutic prospects.Orphanet J Rare Dis. 2014; 9: 71Crossref PubMed Scopus (41) Google Scholar Mutation, epigenetic changes, and alterations in the expression of mucins have also been observed and prognostically linked in many cancers.12King R.J. Yu F. Singh P.K. Genomic alterations in mucins across cancers.Oncotarget. 2017; 8: 67152-67168Crossref PubMed Google Scholar, 13Yamada N. Kitamoto S. 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Pai R.K. GNAS is frequently mutated in both low-grade and high-grade disseminated appendiceal mucinous neoplasms but does not affect survival.Hum Pathol. 2014; 45: 1737-1743Crossref PubMed Scopus (56) Google Scholar, 20Zauber P. Berman E. Marotta S. Sabbath-Solitare M. Bishop T. Ki-ras gene mutations are invariably present in low-grade mucinous tumors of the vermiform appendix.Scand J Gastroenterol. 2011; 46: 869-874Crossref PubMed Scopus (38) Google Scholar, 21Pai R.K. Hartman D.J. Gonzalo D.H. Lai K.K. Downs-Kelly E. Goldblum J.R. Liu X. Patil D.T. Bennett A.E. Plesec T.P. Kuan S.F. Nikiforova M.N. Shadrach B. Pai R.K. Serrated lesions of the appendix frequently harbor KRAS mutations and not BRAF mutations indicating a distinctly different serrated neoplastic pathway in the appendix.Hum Pathol. 2014; 45: 227-235Crossref PubMed Scopus (37) Google Scholar Mutation of TP53 appears to be associated with high-grade disease,16Noguchi R. Yano H. Gohda Y. Suda R. Igari T. Ohta Y. Yamashita N. Yamaguchi K. Terakado Y. Ikenoue T. Furukawa Y. Molecular profiles of high-grade and low-grade pseudomyxoma peritonei.Cancer Med. 2015; 4: 1809-1816Crossref PubMed Scopus (45) Google Scholar, 17Nummela P. Saarinen L. Thiel A. Järvinen P. Lehtonen R. Lepistö A. Järvinen H. Aaltonen L.A. Hautaniemi S. Ristimäki A. Genomic profile of pseudomyxoma peritonei analyzed using next-generation sequencing and immunohistochemistry.Int J Cancer. 2015; 136: E282-E289Crossref PubMed Scopus (56) Google Scholar and immunohistochemical overexpression of p53 portends a worse prognosis.16Noguchi R. Yano H. Gohda Y. Suda R. Igari T. Ohta Y. Yamashita N. Yamaguchi K. Terakado Y. Ikenoue T. Furukawa Y. Molecular profiles of high-grade and low-grade pseudomyxoma peritonei.Cancer Med. 2015; 4: 1809-1816Crossref PubMed Scopus (45) Google Scholar, 22Shetty S. Thomas P. Ramanan B. Sharma P. Govindarajan V. Loggie B. Kras mutations and p53 overexpression in pseudomyxoma peritonei: association with phenotype and prognosis.J Surg Res. 2013; 180: 97-103Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Other mutated genes have been found occasionally, including AKT1, APC, ATM, CTNNB1, JAK3, MET, PIK3CA, RB1, SMAD4, and STK11.14Hara K. Saito T. Hayashi T. Yimit A. Takahashi M. Mitani K. Takahashi M. Yao T. A mutation spectrum that includes GNAS, KRAS and TP53 may be shared by mucinous neoplasms of the appendix.Pathol Res Pract. 2015; 211: 657-664Crossref PubMed Scopus (31) Google Scholar, 17Nummela P. Saarinen L. Thiel A. Järvinen P. Lehtonen R. Lepistö A. Järvinen H. Aaltonen L.A. Hautaniemi S. Ristimäki A. Genomic profile of pseudomyxoma peritonei analyzed using next-generation sequencing and immunohistochemistry.Int J Cancer. 2015; 136: E282-E289Crossref PubMed Scopus (56) Google Scholar, 23Liu X. Mody K. de Abreu F.B. Pipas J.M. Peterson J.D. Gallagher T.L. Suriawinata A.A. Ripple G.H. Hourdequin K.C. Smith K.D. Barth Jr., R.J. Colacchio T.A. Tsapakos M.J. Zaki B.I. Gardner T.B. Gordon S.R. Amos C.I. Wells W.A. Tsongalis G.J. Molecular profiling of appendiceal epithelial tumors using massively parallel sequencing to identify somatic mutations.Clin Chem. 2014; 60: 1004-1011Crossref PubMed Scopus (68) Google Scholar One study reported co-amplification of MCL1 and JUN in a subset of PMP.24Sio T.T. Mansfield A.S. Grotz T.E. Graham R.P. Molina J.R. Que F.G. Miller R.C. Concurrent MCL1 and JUN amplification in pseudomyxoma peritonei: a comprehensive genetic profiling and survival analysis.J Hum Genet. 2014; 59: 124-128Crossref PubMed Scopus (25) Google Scholar Important differences exist between the genetic abnormalities observed in appendiceal and colorectal neoplasia.25Levine E.A. Blazer III, D.G. Kim M.K. Shen P. Stewart IV, J.H. Guy C. Hsu D.S. Gene expression profiling of peritoneal metastases from appendiceal and colon cancer demonstrates unique biologic signatures and predicts patient outcomes.J Am Coll Surg. 2012; 214: 599-606Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar GNAS mutations are rare in the colorectum, although common not only in appendiceal mucinous neoplasia but also in intraductal papillary mucinous neoplasm of the pancreas,15Nishikawa G. Sekine S. Ogawa R. Matsubara A. Mori T. Taniguchi H. Kushima R. Hiraoka N. Tsuta K. Tsuda H. Kanai Y. Frequent GNAS mutations in low-grade appendiceal mucinous neoplasms.Br J Cancer. 2013; 108: 951-958Crossref PubMed Scopus (120) Google Scholar, 26Hosoda W. Sasaki E. Murakami Y. Yamao K. Shimizu Y. Yatabe Y. GNAS mutation is a frequent event in pancreatic intraductal papillary mucinous neoplasms and associated adenocarcinomas.Virchows Arch. 2015; 466: 665-674Crossref PubMed Scopus (57) Google Scholar which itself can occasionally produce PMP. In the colorectum, the commonest oncogenic pathway is APC mutation, leading to abnormalities in the Wnt signaling pathway.27Fearon E.R. Molecular genetics of colorectal cancer.Annu Rev Pathol. 2011; 6: 479-507Crossref PubMed Scopus (1224) Google Scholar In the appendix, mutant APC appears to be less common, which is consistent with the finding that nuclear β-catenin expression is seen in a relatively small proportion of appendiceal adenocarcinomas.28Chu P.G. Chung L. Weiss L.M. Lau S.K. Determining the site of origin of mucinous adenocarcinoma: an immunohistochemical study of 175 cases.Am J Surg Pathol. 2011; 35: 1830-1836Crossref PubMed Scopus (79) Google Scholar Furthermore, even though many mucinous appendiceal tumors show serrated features morphologically, they rarely show the microsatellite instability, loss of expression of DNA mismatch repair proteins, and BRAF mutations typical of the serrated pathway in the colorectum.17Nummela P. Saarinen L. Thiel A. Järvinen P. Lehtonen R. Lepistö A. Järvinen H. Aaltonen L.A. Hautaniemi S. Ristimäki A. Genomic profile of pseudomyxoma peritonei analyzed using next-generation sequencing and immunohistochemistry.Int J Cancer. 2015; 136: E282-E289Crossref PubMed Scopus (56) Google Scholar, 29Misdraji J. Burgart L.J. Lauwers G.Y. Defective mismatch repair in the pathogenesis of low-grade appendiceal mucinous neoplasms and adenocarcinomas.Mod Pathol. 2004; 17: 1447-1454Crossref PubMed Scopus (32) Google Scholar, 30Taggart M.W. Galbincea J. Mansfield P.F. Fournier K.F. Royal R.E. Overman M.J. Rashid A. Abraham S.C. High-level microsatellite instability in appendiceal carcinomas.Am J Surg Pathol. 2013; 37: 1192-1200Crossref PubMed Scopus (29) Google Scholar Next-generation sequencing (NGS) methods, including whole-exome sequencing (WES), have been shown to be valuable tools in cancer genetics, allowing the direct sequencing of all genes within a sample. Paired analyses using both tumor-derived and nontumor samples can allow for the powerful identification of somatic variants and for approximation of the mutation frequencies within the sample.31Van Allen E.M. Wagle N. Stojanov P. Perrin D.L. Cibulskis K. Marlow S. et al.Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine.Nat Med. 2014; 20: 682-688Crossref PubMed Scopus (408) Google Scholar In this study, we have applied WES to paired PMP samples from five individuals, investigating the genetic profiles of the tumors. Five formalin-fixed, paraffin-embedded samples were included in this study, all being histologically confirmed low-grade mucinous carcinoma peritonei of appendiceal origin. The patients were randomly selected from patients who had cytoreductive surgery with hyperthermic intraperitoneal chemotherapy within a 12-month window. Ethical approval for the study was obtained from the National Research Ethics Service, reference 09/H0504/3. Histologic slides from each case were retrieved from the laboratory archives and were reviewed to confirm the diagnosis of low-grade mucinous carcinoma peritonei according to criteria from the Peritoneal Surface Oncology Group International (Figure 1).10Carr N.J. Cecil T.D. Mohamed F. Sobin L.H. Sugarbaker P.H. González-Moreno S. Taflampas P. Chapman S. Moran B.J. Peritoneal Surface Oncology Group InternationalA consensus for classification and pathologic reporting of pseudomyxoma peritonei and associated appendiceal neoplasia: the results of the Peritoneal Surface Oncology Group International (PSOGI) modified Delphi process.Am J Surg Pathol. 2016; 40: 14-26Crossref PubMed Scopus (375) Google Scholar Patients with lesions classified as high-grade mucinous carcinoma peritonei or who had previous adjuvant chemotherapy, lymph node metastasis, and PMP from other primary sites were excluded from the study. Tissue sections cut at 10-μm thickness were stained with cresyl violet, dehydrated in ethanol, and air dried. Laser-capture microdissection to select neoplastic cells was performed with a Leica AS LMD (Leica Microsystems, Wetzlar, Germany), and captured tissue was collected directly into lysis buffer. Nonmalignant material was similarly isolated from the slides for use as a normal comparator for tumor-normal–paired somatic variant calling. Whole-exome enrichment for the tumor and normal samples was performed with SureSelect Human All Exon kits (Agilent, Santa Clara, CA). The V5 kit was used for the PMP220 sample pair, and V6 was used for all others. This change was because of the availability of the updated product after the initial pilot work with PMP220. For all samples, sequencing was performed with a HiSeq 2500 (Illumina, San Diego, CA), with an approximately twofold more sequence data being generated for tumor samples than for normal samples to improve somatic mutation detection sensitivity. NGS reads were aligned to GRCh37 (hg19) with the use of Burrows-Wheeler Aligner version 0.7.12 (https://sourceforge.net/projects/bio-bwa).32Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26655) Google Scholar Paired somatic variant calling was performed with VarScan 2 version 2.3.6 (https://github.com/dkoboldt/varscan),33Koboldt D.C. Zhang Q. Larson D.E. Shen D. McLellan M.D. Lin L. Miller C.A. Mardis E.R. Ding L. Wilson R.K. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing.Genome Res. 2012; 22: 568-576Crossref PubMed Scopus (2942) Google Scholar assuming a tumor sample purity of 50% and requiring a minimum mutation frequency of 1% in the tumor samples. Candidate genes were collated according to the gene content of the TruSeq Amplicon – Cancer Panel (Illumina) as well as the top 20 mutated genes reported for PMP within the COSMIC database version 81,34Forbes S.A. Beare D. Boutselakis H. Bamford S. Bindal N. Tate J. Cole C.G. Ward S. Dawson E. Ponting L. Stefancsik R. Harsha B. Kok C.Y. Jia M. Jubb H. Sondka Z. Thompson S. De T. Campbell P.J. COSMIC: somatic cancer genetics at high-resolution.Nucleic Acids Res. 2017; 45: D777-D783Crossref PubMed Scopus (1253) Google Scholar in addition to the three most commonly hyperexcreted mucins.11Amini A. Masoumi-Moghaddam S. Ehteda A. Morris D.L. Secreted mucins in pseudomyxoma peritonei: pathophysiological significance and potential therapeutic prospects.Orphanet J Rare Dis. 2014; 9: 71Crossref PubMed Scopus (41) Google Scholar The 54 candidate genes are presented in Supplemental Table S1. Variants of interest were manually assessed in the raw data to minimize false-positive calls because of artifacts. The mutational signature of the tumors was investigated to try to gain insight into the mutation mechanisms acting within the tumors. The mutation signature was defined as the distribution of mutations within the triplet nucleotide context, this being the mutated nucleotide with the 5′ and 3′ adjacent nucleotides.35Alexandrov L.B. Nik-Zainal S. Wedge D.C. Aparicio S.A. Behjati S. Biankin A.V. et al.Signatures of mutational processes in human cancer.Nature. 2013; 500: 415-421Crossref PubMed Scopus (6224) Google Scholar Regions of loss of heterozygosity (LOH) were assessed with B allele frequency (BAF) segmentation version 1.2 (https://emea.illumina.com/techniques/microarrays/array-data-analysis-experimental-design/genomestudio.html).36Staaf J. Lindgren D. Vallon-Christersson J. Isaksson A. Göransson H. Juliusson G. Rosenquist R. Höglund M. Borg A. Ringnér M. Segmentation-based detection of allelic imbalance and loss-of-heterozygosity in cancer cells using whole genome SNP arrays.Genome Biol. 2008; 9: R136Crossref PubMed Scopus (115) Google Scholar Inherent to this approach is the default assumption that, for a given heterozygous variant, the two alleles will be present within a sample in equal proportions, which is quantified with the BAF, in the case of a heterozygote therefore expected to be 0.5. In the case of an LOH event, alleles on the chromosomal segment affected will be skewed, with one allele being overrepresented within the NGS reads. BAF segmentation considers this skewing effect across regions that contain multiple variants, arriving at a consensus decision as to whether a region has undergone LOH. This analysis was performed by considering only variants that have a read depth of >50 times in both the paired tumor and normal samples for a participant to improve the accuracy of allelic ratio determination. LOH regions were manually curated to maximize accuracy. Clinical and pathologic details for the five recruited individuals are detailed in Table 1, with representative histologic findings shown in Figure 1. LAMN was the primary lesion in all cases; case PMP248 had received a prior appendectomy because of PMP elsewhere, and review of the slides from the referring hospital confirmed LAMN. In all cases there was extension of neoplastic cells and mucin beyond the right lower quadrant of the abdomen. The intraabdominal disease in PMP is a malignant condition appropriately termed mucinous carcinoma peritonei; in all of the studied cases the peritoneal disease was classified as low grade.10Carr N.J. Cecil T.D. Mohamed F. Sobin L.H. Sugarbaker P.H. González-Moreno S. Taflampas P. Chapman S. Moran B.J. Peritoneal Surface Oncology Group InternationalA consensus for classification and pathologic reporting of pseudomyxoma peritonei and associated appendiceal neoplasia: the results of the Peritoneal Surface Oncology Group International (PSOGI) modified Delphi process.Am J Surg Pathol. 2016; 40: 14-26Crossref PubMed Scopus (375) Google Scholar All patients were alive 53 months to 58 months after surgery, although recurrence of intraabdominal tumor occurred in patient PMP247 32 months after surgery. No patient had lymph node metastases or extra-abdominal disease.Table 1Clinical Details of the Five Patients, Including Levels of Tumor Markers at the Time of DiagnosisClinical detailPMP220PMP228PMP244PMP247PMP248Age, years6535506545SexFemaleFemaleMaleFemaleFemalePrimary lesionLAMNLAMNLAMNLAMNLAMNPCI2821342818Serum CEA, U/mL3411201514Serum CA 125, U/mL7949137450Serum CA 19-9, U/mL1222337389139Follow-up, months5856535353CA 19-9, cancer antigen 19-9 (reference range, 0 to 33 U/mL); CA 125, cancer antigen 125 (reference range, 0 to 35 U/mL); CEA, carcinoembryonic antigen (reference range, 0 to 5 U/mL); LAMN, low-grade appendiceal mucinous neoplasm; PCI, peritoneal cancer index (range of possible scores = 0 to 39); PMP, pseudomyxoma peritonei. Open table in a new tab CA 19-9, cancer antigen 19-9 (reference range, 0 to 33 U/mL); CA 125, cancer antigen 125 (reference range, 0 to 35 U/mL); CEA, carcinoembryonic antigen (reference range, 0 to 5 U/mL); LAMN, low-grade appendiceal mucinous neoplasm; PCI, peritoneal cancer index (range of possible scores = 0 to 39); PMP, pseudomyxoma peritonei. NGS data generation for the paired samples yielded data of sufficient quality for downstream analysis for all samples; coverage of candidate genes are detailed in Supplemental Table S1. An average read depth across the exome capture region was 65.7× and 112.7× for the normal and tumor samples, respectively, with ≥90% of the target exome regions covered in all cases. Variant calling generated 112 to 173 coding somatic mutation calls in each of the samples (Table 2). Across the five samples, 10% of the identified coding mutations were frameshifts, and a further 3% were stopgain and were therefore expected to be protein truncating. Twenty-six percent of the coding mutations were possible effecters of splicing (ie, being within 10 nucleotides of the intron–exon boundary) and 43% were nonsynonymous. The final 18% of the mutations were synonymous. There was a substantial excess of C>T transitions in the context of N[C>T]G (Supplemental Figure S1). This mutation signature is consistent with deamination of 5-methylcytosine as the molecular mutation mechanism and is a common mutational mechanism in many cancers.35Alexandrov L.B. Nik-Zainal S. Wedge D.C. Aparicio S.A. Behjati S. Biankin A.V. et al.Signatures of mutational processes in human cancer.Nature. 2013; 500: 415-421Crossref PubMed Scopus (6224) Google ScholarTable 2Somatic Coding Variant Call Counts for Each SampleVariantPMP220PMP228PMP244PMP247PMP248Frameshift03291416Nonsynonymous7544715562Splicing753603530Stopgain42446Synonymous2621292628Sum112152173134142PMP, pseudomyxoma peritonei. Open table in a new tab PMP, pseudomyxoma peritonei. To shortlist variants that may be of significance in these samples, two approaches were applied. First, a candidate gene-based analysis that used the 51 candidate genes was applied to ide
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