{"paper_id":"e2514cfd-c36c-45fb-99f7-75e3c5cd645d","body_text":"Epithelial ovarian tumors have historically been treated as a homogenous group in the clinic, despite clear histopathological and molecular data showing that distinct subgroups exist: serous, endometrioid, clear cell and mucinous. High-grade serous and low-grade serous tumors comprise distinct groups, while endometrioid and clear-cell histologies are different again from serous but with some overlapping genetic events. It is now clear that these molecular distinctions reflect differences in site of origin, with high-grade serous tumors now thought to arise from the fallopian tube fimbriae, low-grade serous tumors from the ovarian epithelium, and clear cell and endometrioid tumors arising from endometriosis, which itself is derived from the endometrium. However, the origin of the mucinous group remains controversial. Many mucinous ovarian tumors (MOTs) formerly classified as primary are now recognized to have been mis-diagnosed metastases from predominantly gastrointestinal or endocervical sites. However, some mucinous tumors do appear to be ovarian primaries, particularly benign and borderline tumors, which generally have a good prognosis not consistent with a metastatic tumor. Carcinomas associated with benign and borderline elements and/or with an early-stage, unilateral presentation are also thought to be primary ovarian in origin.\nOur understanding of the genomic landscape of MOTs is limited. Older reports are likely to include a high proportion of metastatic mucinous tumors, and the rarity of true primary mucinous tumors results in limited investigations. Nonetheless, we and others have shown that mucinous tumors have a high proportion of mutations in RAS pathway genes and aberration of  CDKN2A  (p16) [ 1 ,  2 ]. Beyond these common drivers, little is known, and the predominantly stable genomic copy number profiles we have observed in this tumor type [ 3 ] suggest that somatic point mutations are likely to be more relevant. In this study, we have undertaken exome sequencing of a large cohort of MOTs, and have further investigated lead candidates in a validation cohort of 58 cases.\n\nFresh-frozen MOTs were accessed from bio-banked specimens collected and cryopreserved at the time of surgical resection for a primary ovarian tumor, prior to chemotherapy administration. Samples comprised 22 benign cystadenomas, 29 tumors of low malignant potential (herein referred to as borderline tumors) and 31 carcinomas [ 4 ,  5 ]. Hospitals contributing samples between 1993 and 2011 included those in the south of England, UK [ 5 ], and in Australia (Southern Health and the Australian Ovarian Cancer Study [AOCS] [ 4 ]). Blood samples used for germline DNA extraction were also collected prior to surgery. Thorough histological classification was based on the entire specimen at time of diagnosis, although all cases underwent retrospective pathological review using information obtained from the pathology report and histological assessment according to established criteria [ 6 ] in order to exclude likely metastases. Cases were also excluded if there was insufficient tumor epithelium for nucleic acid extraction. Carcinoma grade was derived from the diagnostic pathology report because there were insufficient cases with archival specimens available for re-review. Clinicopathological data are provided in Additional file  1 : Table S1.\nThis study was performed in accordance with the ethical standards of the Peter MacCallum Cancer Centre Human Research Ethics Committee (Approvals 09/29 and 01/38) and all participants provided written informed consent for tissue collection. This study conforms to the Declaration of Helsinki.\nTumor genomic DNA was isolated by needle microdissection of areas with greater than 80 % neoplastic cellularity from consecutive 10 μm hematoxylin and eosin (H&E)-stained tumor sections and extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) as per the recommended protocol. Matched germline DNA was extracted from whole blood (19 cases) or paired uninvolved ovarian stroma (5 cases). Whole genome amplification (WGA) was performed on 20–50 ng of tumor and germline DNA using the Repli-G Phi-mediated amplification system (Qiagen) and the product was used to confirm mutations detected by exome sequencing and to perform candidate gene mutation analysis.\nLibraries were constructed from 500 ng of unamplified tumor or germline DNA following the Illumina TruSeq DNA Sample Preparation procedure (Illumina, San Diego, CA, USA), followed by exome capture using the NimbleGen SeqCap EZ Human Exome Library v1 or v2 capture kit (Roche NimbleGen, Heidelberg, Germany). Each resulting paired-end library was sequenced on one-third of an Illumina HiSeq2000 lane using 75 bp or 100 bp reads. Library preparation and detailed summary statistics for all samples are listed in Additional file  1 : Table S2.\nPurity filtered paired-end reads were quality checked with FastQC (v0.10.1) and trimmed for low quality bases and adaptor if necessary using Cutadapt (v1.1). Reads were then aligned to the human genome (GRCh37/hg19) using BWA-MEM (v0.7.7-r441). Duplicates were marked using Picard (v1.77) followed by local insertion-deletion (indel) re-alignment and base quality score recalibration using GATK (v2.7-2-g6bda569). Somatic single nucleotide variants (SNVs) and indels were called using the following algorithms with the matched germline data used as reference: MuTect (v2.7-1-g42d771f), JointSNVMix (v0.8-b2) and Somatic Sniper (1.0.2.2-1-g8ee3999) (SNVs only); SomaticIndelDetector (v1.0.4905) (indels only); and VarScan (v2.3.4) (both SNVs and indels). In addition, Pindel (v0.2.5a3) and GATK Unified Genotyper (v2.7-2-g6bda569) were used to call SNVs and indels separately in the tumor and germline samples.\nInitial variant predictions were filtered to require that (1) SNVs were called by ≥2 of MuTect, JointSNVMix, Somatic Sniper, VarScan or Unified Genotyper, (2) indels were called by any of SomaticIndelDetector, VarScan, Pindel or Unified Genotyper, (3) the variant was present in ≥10 reads in the tumor (Pindel) or ≥2 reads in the tumor (all other callers), (4) the mutant allele frequency was ≤5 % in the matched germline sample, and (5) the mutant allele fraction was at least 10 % higher in the tumor than in the matched germline sample for indels called by Pindel and Unified Genotyper. Finally, any remaining germline single nucleotide polymorphisms (SNPs) or common sequence artifacts were eliminated by requiring that the variant allele was not observed in more than two of the other germline samples from this cohort or more than two (of 147) in-house germline exome sequences [ 7 ], and had an Exome Variant Server (ESP6500 SI-v2) minor allele frequency of ≤5 %.\nPredicted somatic mutations were annotated with Ensembl v73 information and those with impact predictions overlapping coding regions and splice sites (±2 bp) were considered for further analysis. Due to restrictions of our ethics approval, we are not able to provide BAM files; however, all variants are available in Additional file  1 : Table S3. All coding mutations were manually reviewed by examination of BAM files using the Integrative Genomics Viewer.\nSelected somatic mutations were independently assessed by polymerase chain reaction (PCR) and Sanger sequencing of the tumor DNA as described previously [ 8 ]. Somatic status was confirmed by also resequencing the corresponding germline sample.\nThirty-two known somatic mutations in  KRAS ,  BRAF ,  TP53  and  CDKN2A  that had been independently validated in other studies of this cohort by Sanger sequencing were used to assess the sensitivity of somatic mutation calling, with 96.9 % known somatic variants successfully identified (27/27 SNVs and 4/5 indels). Failure to identify a known 36 bp complex indel in  CDKN2A  was complicated by low read depth owing to high GC content for this gene; this variant was included in subsequent analyses. The confirmation rate of novel variants by Sanger sequencing was 93.6 % (208/223 SNVs and 25/26 indels).\nThe MuSiC algorithm (v0.4) was applied using default parameters to identify genes significantly enriched for mutations, given sequence type, context and estimated background rate [ 9 ]. Genes mutated in two or more samples with a  P -value ≤0.05 at a false discovery rate of ≤0.1 in any of the three tests were considered significant. Furthermore, OncodriveFM (accessed through the online IntOGen platform at [ 10 ]) was used to identify genes with significant bias towards the accumulation of functional mutations ( P -value ≤0.05 and  q -value ≤0.1) [ 11 ,  12 ].\nThe complete coding exons of  ELF3  (exons 2–9) and  ERBB3  (exons 1–28) were assessed by direct Sanger sequencing using the primers listed in Additional file  1 : Table S4. To assess the  ELF3  c.1001 + 1_1001 + 2insGG mutation on mRNA splicing, cDNA amplification and direct sequencing were performed using primers listed in Additional file  1 : Table S4. Targeted Sanger sequencing of mutation hotspots in  TP53  (exons 4–9),  BRAF  (codon 600),  KRAS / HRAS / NRAS  (codons 12, 13 and 61) and the coding region of  CDKN2A  (exons 1–3) were sequenced using primers previously described [ 2 ]. Somatic mutations identified in these genes have previously been published for a subset of the benign and borderline mucinous tumors [ 2 ].\nHigh-resolution melt analysis was used to screen for mutations at hotspot codon 201 of  GNAS  and in the coding exons of  KLF5  (exons 1–4). For this, 15 ng WGA tumor DNA was amplified in duplicate using the primers listed in Additional file  1 : Table S4, followed by melt analysis on the LightCycler 480 Instrument using Gene Scanning Software (Roche). Samples with variant melt curves in duplicate PCRs were independently amplified using the same primers ( KLF5 ) or an independent primer set ( GNAS ) and Sanger sequenced to confirm sequence variations.\nHER2 status was ascertained based on the detection of high-level gene amplification by high-density genome-wide SNP arrays (Affymetrix, Santa Clara, CA, USA) (35 cases) [ 2 ,  3 ], SNP array data plus immunohistochemistry (IHC) (23 cases), or by IHC alone (18 cases). For 16 cases, HER2 IHC was evaluated on 4 μM formalin-fixed paraffin-embedded whole sections using anti-HER2 antibody clone 4B5 (Ventana Medical Systems, Tuscon, AZ, USA). Staining was scored visually according to standard guidelines [ 13 ]; briefly, an IHC score of 3+ (strong uniform membrane staining of >30 % tumor cells) was categorized as HER2 positive; equivocal cases (score of 2+, strong complete membrane staining in <30 % tumor cells or weak to moderate heterogeneous staining in >10 % tumor cells) were only considered HER2 positive if accompanied by array-based copy number amplification of the  ERBB2  locus. An equivocal score without amplification confirmation, and tumors that scored 0 or 1 (no staining or weak incomplete membrane staining in any proportion of tumor cells), were considered negative. For the remaining 25 cases, the HER2 IHC score was derived from Anglesio et al. [ 1 ], who used comparable classification guidelines.\n\nTo profile the somatic mutation spectrum of mucinous tumors of the ovary, we performed whole exome sequencing on 24 tumors including 5 benign cystadenomas, 8 borderline tumors and 11 carcinomas (Table  1 , Additional file  1 : Table S1). A mean coverage depth of 144× was achieved in both neoplastic and non-cancerous specimens (range 53-fold to 224-fold) and 91 % of the bases were covered by at least 20 uniquely mapping reads (Additional file  1 : Table S2). Using stringent criteria, 1126 somatic coding and essential splice site mutations were identified (1031 SNVs and 95 indels), of which 841 were predicted to alter protein sequence (Additional file  1 : Table S3). These included 44 (5.2 %) nonsense, 60 (7.1 %) frameshift indel, 16 (1.9 %) splice site, 27 (3.2 %) inframe indel and 694 (82.5 %) missense mutations. Benign and borderline tumors had on average 25.4 (range 21–38) and 32.9 (range 2–76) coding mutations per tumor, equating to a frequency of 0.8 mutations/Mb and 0.9 mutations/Mb respectively. Although variable, this mutation burden did not differ between benign and borderline tumors but was significantly lower when compared to the carcinomas (average of 66.9 mutations per sample and 1.5 mutations/Mb) ( P  = 0.008 vs. benign and  P  = 0.047 vs. borderline) attributed mostly to an accumulation of missense mutations in the carcinomas (Fig.  1a , Additional file  1 : Table S5). There were no hyper-mutated cases (defined as >10 mutations/Mb) indicative of a mutator phenotype such as mismatch repair deficiency. Relative to other cancer types, MOTs showed a similar somatic mutation density to breast, serous ovarian and pancreatic cancers, but a lower density than colorectal and stomach tumors [ 14 ,  15 ]. Table 1 Cohort summary Clinical feature Discovery (n = 24) Validation (n = 58) Type:  Benign 5 17  Borderline 8 21  Carcinoma 11 20 Grade (carcinoma)  1 5 6  2 3 10  3 2 4  Not known 1 Stage (carcinoma)  1 7 16  2 1 1  3 1 1  Not known 2 2 Age (average ± standard deviation)  Benign 49.8 ±5.9 59.5 ±11.3  Borderline 55.4 ±15.8 53.6 ±16.0  Carcinoma 62.3 ±11.0 53.8 ±11.1 Laterality  Unilateral 21 54  Bilateral 2 4  Not known 1 Size  <10 cm 2 1  ≥10 cm 21 57  Not known 1 0 Fig. 1 Mutational landscape of MOTs identified by exome sequencing. Samples are grouped according to pathological classification and ordered from lowest to highest mutation frequency.  a  Somatic mutation frequency (left Y-axis) and number of coding mutations by consequence (right Y-axis).  b  Relative frequency of somatic mutations according to base substitution type. Substitutions were categorized by the six possible base-pair changes\nCohort summary\nMutational landscape of MOTs identified by exome sequencing. Samples are grouped according to pathological classification and ordered from lowest to highest mutation frequency.  a  Somatic mutation frequency (left Y-axis) and number of coding mutations by consequence (right Y-axis).  b  Relative frequency of somatic mutations according to base substitution type. Substitutions were categorized by the six possible base-pair changes\nThe mutation spectrum was dominated by C>T transitions, comprising 63.9 % of somatic substitutions, and this was common to all three tumor subtypes (Fig.  1b ). Mutations in this context demonstrated a marked preference for NpCpG trinucleotides (Additional file  2 , Figure S1a), the optimum motif for spontaneous 5-methylcytosine deamination [ 16 ]. An equivalent signature is frequently seen in other epithelial tumors of the gastrointestinal tract, but is different to that observed in other cancers of the female reproductive system including high-grade serous ovarian carcinoma (Additional file  2 : Figure S1b). Taken together these findings are consistent with MOTs having a shared lineage distinct from that of other ovarian epithelial tumors.\nProtein-altering mutations were detected in 761 genes, of which 42 were mutated in two or more of the 24 tumors. Among the most frequently mutated were known mucinous ovarian cancer genes  KRAS ,  BRAF  and  CDKN2A  (Table  2 , Fig.  2 ). Interestingly,  TP53  was the second most frequently mutated gene, with seven mutations identified. Eight genes were significantly mutated based on a statistically significant accumulation of mutations by both MuSiC [ 9 ] and OncodriveFM [ 11 ] (Table  2 ). Other genes predicted by one algorithm were also notable; for example,  ERBB3  (MuSiC) and  GNAS  and  FBXW7  (OncodriveFM) (Table  2 ). Based on these predictions and observation in other cancer types, five novel candidate drivers not previously reported in MOTs were selected for validation in an independent cohort:  TP53  (7/24),  ELF3  (3/24),  ERBB3  (2/24),  GNAS  (2/24) and  KLF5  (2/24) (Table  2 , Fig.  2 , Additional file  1 : Table S6). Our validation study of the tumor suppressor gene  RNF43  has been published previously [ 8 ]. In addition, known cancer genes for this ovarian subtype were evaluated in parallel to assess their relationship with new drivers including HER2 (by immunohistochemistry and copy number analysis) and mutations in  KRAS ,  BRAF ,  CDKN2A  and other RAS pathway members  NRAS  and  HRAS . Table 2 Candidate driver genes with significantly recurrent somatic mutations in mucinous ovarian tumors Exome cohort SMG prediction Validation cohort Overall Gene Mutated samples Nonsense, frameshift indel, splice Inframe indel, missense OncodriveFM MuSiC  q -value Mutated samples Nonsense, frameshift indel, splice Inframe indel, missense Mutated samples \n q -value FCPT LRT CT \n KRAS \n 12 0 12 1.34 × 10 −13 \n 0 0 0 32 0 33 44/82 \n TP53 \n 7 1 6 3.66 × 10 −11 \n 2.86 × 10 −7 \n 1.41 × 10 −9 \n 1.17 × 10 −12 \n 15 0 15 22/82 \n BRAF \n 6 0 6 2.45 × 10 −8 \n 7.77 × 10 −7 \n 0 6.18 × 10 −12 \n 4 0 5 10/82 \n CDKN2A \n 5 5 2 0.0043 1.20 × 10 −10 \n 0 7.93 × 10 −17 \n 5 5 0 10/63 \n RNF43 \n 5 5 0 4.65 × 10 −6 \n – 0.0009 0.0004 3 2 1 8/65 b \n \n ELF3 \n 3 2 1 0.0079 – 0.0003 0.0004 1 1 0 4/65 \n ARID1A \n 2 2 0 0.0164 – 0.0933 – – – – 2/24 \n DCLK1 \n 2 0 2 – – 0.0569 – – – – 2/24 \n ERBB3 \n 2 0 3 – – 0.0014 0.0374 0 0 0 2/43 \n FBXW7 \n 2 1 1 0.0207 – – – – – – 2/24 \n GNAS \n a \n 2 0 2 9.05 × 10 −8 \n – – – 3 0 3 5/81 \n KLF5 \n 2 2 0 0.0164 – 0.0056 0.0536 0 0 0 2/43 \n LPHN3 \n 2 0 2 0.0493 – – – – – – 2/24 \n LRRK2 \n 2 1 1 0.0997 – – – – – – 2/24 \n TTF1 \n 2 0 2 – – 0.0569 – – – – 2/24 All non-synonymous mutations in listed genes were validated by Sanger sequencing \n CT  convolution test,  FCPT  Fisher’s combined  P -value test,  LRT  likelihood ratio test,  SMG  significantly mutated gene \n a Only mutations involving the hotspot codon 201 are reported \n b Includes samples from Ryland et al. [ 8 ] plus 16 additional samples Fig. 2 Candidate driver genes in MOTs. Significantly mutated genes identified by OncodriveFM and MuSiC analyses are arranged vertically by their frequency of mutated samples in the whole exome sequencing data.  Color  indicates mutation consequence. Selected genes were also investigated in a validation cohort of mucinous tumors. Each  column  denotes an individual tumor (ordered as listed in Additional file  1 : Table S1), which have been arranged to emphasize mutational groups. Genomic aberrations in other MAPK pathway genes were also screened for mutations.  LOH  loss of heterozygosity\nCandidate driver genes with significantly recurrent somatic mutations in mucinous ovarian tumors\nAll non-synonymous mutations in listed genes were validated by Sanger sequencing\nCT  convolution test,  FCPT  Fisher’s combined  P -value test,  LRT  likelihood ratio test,  SMG  significantly mutated gene\na Only mutations involving the hotspot codon 201 are reported\nb Includes samples from Ryland et al. [ 8 ] plus 16 additional samples\nCandidate driver genes in MOTs. Significantly mutated genes identified by OncodriveFM and MuSiC analyses are arranged vertically by their frequency of mutated samples in the whole exome sequencing data.  Color  indicates mutation consequence. Selected genes were also investigated in a validation cohort of mucinous tumors. Each  column  denotes an individual tumor (ordered as listed in Additional file  1 : Table S1), which have been arranged to emphasize mutational groups. Genomic aberrations in other MAPK pathway genes were also screened for mutations.  LOH  loss of heterozygosity\nWe and others have previously described the importance of the RAS pathway and p16 in MOTs [ 1 – 3 ]. Here we extended this analysis and found 56 cases with mutations in  KRAS ,  BRAF  and  NRAS  (68.3 %).  BRAF  mutations were significantly more prevalent in the carcinomas (7/31, 22.6 %) than in the borderline (3/29, 10.3 %) or benign tumors (0/22,  P  = 0.036 Fisher’s exact test), suggesting an association with a more aggressive phenotype. An alternative mechanism for activation of the MAPK pathway was identified through mutation of the  ras -like gene  RRAS2  in one benign tumor that was  KRAS / BRAF  wild type (Fig.  2 ). We previously reported  RRAS2  gene amplification in this sample [ 2 ]; consistent with this, the Sanger sequencing validation confirmed homozygous amplification of the mutant allele (Additional file  2 : Figure S2). This 9 bp duplication, resulting in reiteration of Gly-Gly-Gly (codons 22–24), occurs in the region of  RRAS2  that is complementary to codons 11–13 within the G1 phosphate-binding loop of conventional ras proteins (P-loop, amino acids 10–17). Interestingly, rare reports of comparable events appear in the literature. Huang et al .  [ 17 ] described a three amino acid  RRAS2  duplication (Gly24_26dup) in the human uterine leiomyosarcoma cell line ST-UT-1; this mutation resulted in enhanced GTP-binding and conferred transforming activity  in vitro . Similarly, in  KRAS , 9 bp and 12 bp tandem repeats of codons 10–12 and 10–13 respectively were identified as an alternative mechanism for  KRAS  oncogenic activation in 2 of 18 chemically induced rat renal mesenchymal tumors [ 18 ]. Triple residue insertions in the P-loop of  HRAS  also demonstrated increased preference for GTP-binding and increased interactions with downstream Raf kinase compared to wild type [ 19 ]. Taken together, these observations indicate that although the  RRAS2  duplication described in this study is an unconventional mutation for  ras  proto-oncogene activation, it is predicted to result in up-regulated MAPK pathway activity.\nA previous study found  KRAS  mutation and  HER2  amplification to be almost mutually exclusive [ 1 ]. Although the number of cases we studied was smaller, we did not see this exclusivity: 2/6 HER2+ borderline and 3/6 HER2+ carcinomas also carried  KRAS  mutations. One caveat to this observation is that HER2 status in this study was based on IHC and/or high-level amplification (SNP array analysis) rather than a combined score including chromogenic in situ hybridization.\nIn addition to the seven somatic  TP53  mutations identified by exome sequencing, Sanger sequencing of the DNA binding domain (exons 4–9) in the validation cohort identified a further 15 mutations at an overall frequency of 22/82 (26.8 %) MOTs, of which 21 were missense mutations (Table  2 , Fig.  2 ). All 22 mutations have been previously reported in a somatic context (IARC  TP53  mutation database release 17). There was a significant difference in  TP53  mutation frequency among the three tumor subtypes ( P  = 0.003, Chi-square test). While there was a similar frequency of  TP53  mutations in benign (2/22, 9.1 %) and borderline (4/29, 13.8 %) tumors, 16/31 (51.6 %) of carcinomas harbored a  TP53  mutation ( P  = 0.002 and  P  = 0.002 compared to benign and borderline tumors respectively, Fisher’s exact test), suggesting that aberrant p53 contributes to the invasive phenotype in a proportion of these ovarian cancers. Both low-grade and high-grade carcinomas harbored mutations, which trended towards increasing frequency with grade (45.5 %, 53.8 % and 66.7 % in Grades 1, 2 and 3 respectively), and with an overall frequency similar to that of gastrointestinal mucinous carcinomas (Additional file  2 : Figure S3). While it is well accepted that  TP53  mutation is an obligatory event in the genesis of high-grade serous ovarian carcinoma, we show by direct sequencing that mutant p53 is also common in mucinous-type ovarian carcinomas, but is a late event in their molecular progression. Interestingly, this group does not share the widespread genomic instability that typifies high-grade serous carcinomas that is contributed to, at least in part, by mutant  TP53 , suggesting different p53 activity in these two contexts.\nThree mutations in the epithelial-specific ETS transcription factor  E74-like factor 3  ( ELF3 ) were detected in three tumors by exome sequencing.  ELF3  was significantly mutated above background (MuSiC) and had an excess of likely deleterious mutations (OncodriveFM) including two frameshift insertions (p.Val345Glyfs*126, p.Asp239Glyfs*62) and a missense substitution (p.Met324Val) (Table  2 , Figs.  2  and  3 ). Sequencing of the coding regions in the expanded cohort identified an additional splice site mutation in a borderline tumor (c.1001 + 1_1001 + 2insGG). Although  ELF3  is thus infrequently mutated (6.9 % borderline tumors and 6.5 % carcinomas), the shared characteristics of the four heterozygous mutations is indicative of a pathogenic role. Three of the mutations are overtly deleterious, including two frameshift indels and a canonical splice site mutation, while the missense mutation is predicted to be deleterious by computational analyses [ 20 – 22 ]. We further investigated the exon 8 splice donor site mutation by cDNA sequencing, which confirmed the use of an alternative donor splice sequence in the mutant allele (Additional file  2 : Figure S4) consistent with the in silico prediction [ 23 ]. This mutation would result in out-of-frame, continued translation into the 3′-untranslated region (p.Tyr335Glyfs*113). cDNA sequencing of this and the two other truncating mutations found that all three mutations were readily detected in the tumor RNA, indicating that these mutations are not the subject of strong nonsense-mediated decay (Additional file  2 : Figure S4). Truncating mutations in this epithelial-specific transcription factor have recently been reported in other cancers, including cancer of the cervix, stomach and bladder [ 24 – 26 ]. Interestingly,  ELF3 -mutated cervical carcinomas express ELF3 at a higher level compared to wild-type tumors [ 24 ]. This result may suggest that both copies of this gene are required and mutation of one allele results in up-regulation of the gene in an attempt to compensate. Alternatively,  ELF3  mutations may only have a selective advantage in tumors highly expressing ELF3. Fig. 3 Distribution of somatic mutations identified in novel significantly mutated genes.  ELF3 ,  KLF5 ,  GNAS  and  ERBB3  are shown in the context of protein domains as predicted by UniProt, with somatic mutations identified in the exome ( closed circle ) and validation ( open circle ) cohorts mapped to each gene.  I-IV  extracellular domains I, II, III and IV,  AT hook & NLS  AT-hook domain and nuclear localization signal,  C2H2  zinc-finger C2H2 domain,  ETS  DNA binding domain,  GTP  GTP nucleotide binding region,  PNT  pointed domain,  SAR  serine-rich and aspartic acid-rich domain,  TAD  transactivation domain,  TKD  tyrosine kinase domain\nDistribution of somatic mutations identified in novel significantly mutated genes.  ELF3 ,  KLF5 ,  GNAS  and  ERBB3  are shown in the context of protein domains as predicted by UniProt, with somatic mutations identified in the exome ( closed circle ) and validation ( open circle ) cohorts mapped to each gene.  I-IV  extracellular domains I, II, III and IV,  AT hook & NLS  AT-hook domain and nuclear localization signal,  C2H2  zinc-finger C2H2 domain,  ETS  DNA binding domain,  GTP  GTP nucleotide binding region,  PNT  pointed domain,  SAR  serine-rich and aspartic acid-rich domain,  TAD  transactivation domain,  TKD  tyrosine kinase domain\nELF3  has previously been identified as a candidate cancer gene; however, its role appears to be context dependent, in keeping with the tissue-specific nature of its transcriptional target genes. An oncogenic role has been suggested for breast cancer, with the gene being amplified and overexpressed [ 27 ,  28 ]. A positive feedback loop between ELF3 and HER2 exists in breast cancer, where ELF3 is both a downstream mediator and activator of HER2 signaling [ 29 ]. Of note in this study, two of two mutated carcinomas were HER2+, while the two mutated borderline tumors were HER2-. However, in a gastrointestinal tissue context, ELF3 may act as a tumor suppressor, because it is involved in positively transcriptionally regulating  TGFBR2 , facilitating the growth inhibitory consequences of TGF-β signaling [ 30 ,  31 ].  ELF3  was identified as a cancer gene in a recent pan-cancer study, with enrichment for mutations in bladder and colorectal cancer [ 32 ]. The frequency of mutations in MOTs suggests that  ELF3  is indeed a cancer gene in this tumor type, but its exact role is unclear from the mutational profile—while the mutations are detrimental in nature, the retention of the wild-type allele argues against a classical tumor suppressor gene functional mechanism.\nAnother transcription factor with a proclivity for truncating mutations was  KLF5 , which encodes a zinc finger transcriptional activator (Table  2 , Figs.  2  and  3 ). Exome sequencing identified two heterozygous frameshift mutations (p.Phe123Leufs*3 and p.Asp238Argfs*16); however, sequencing the coding region in a validation cohort of carcinomas failed to identify additional changes. Collectively,  KLF5  is mutated in 6.7 % (2/30) of mucinous ovarian carcinomas. Like  ELF3 , it has been identified as a pan-cancer gene but enriched for mutations in bladder, colorectal, and head and neck squamous carcinoma [ 32 ], and has been variously described as both an oncogene [ 33 ] and a tumor suppressor gene [ 34 ].\nConsidering exome and validation cohorts, five constitutively activating mutations at arginine codon 201 of the oncogene  GNAS  were identified, including 2/22 (9.1 %) benign cystadenomas, 2/29 (6.9 %) borderline tumors and 1/30 (3.3 %) carcinomas (Table  2 , Figs.  2  and  3 ). Hotspot mutations in this guanine nucleotide-binding protein alpha subunit have recently been identified in other pre-malignant or non-aggressive mucinous-type tumors of gastrointestinal origin, albeit at a higher frequency (Additional file  2 : Figure S3), including intraductal papillary mucinous neoplasm of the pancreas and bile duct [ 35 ,  36 ]; appendiceal mucinous neoplasms (and its associated pseudomyxoma peritonei) [ 37 ,  38 ]; and adenoma of the colon/rectum, stomach and small intestine [ 39 ,  40 ]. In this context, constitutive activation of  GNAS  through codon 201 mutation has been shown to increase levels of cAMP, resulting in prominent mucin production but not cell growth [ 38 ]. Consistent with previous reports, simultaneous  KRAS  mutations were present in four MOTs, although this association was not statistically significant. Thus, unlike gastrointestinal mucinous-type tumors,  GNAS  activation occurs only rarely in those involving the ovary.\nAlthough human epidermal growth factor receptors have been implicated in MOT progression through amplification and overexpression of ERBB2 (HER2), activating mutations in other family members have not been previously described. Exome sequencing identified three  ERBB3  (HER3) mutations (a borderline tumor with concurrent mutations, and a carcinoma) (Table  2 , Figs.  2  and  3 ), including two in the extracellular domain (p.Met91Ile and p.Glu332Lys) and one in the kinase domain (p.Glu925Lys). No additional mutations were identified in a validation screen of 19 carcinomas, giving a final frequency of 4.7 % in MOTs. Frequent  ERBB3  mutations have recently been reported in other cancer types, including those of the colon, gallbladder and stomach [ 41 ,  42 ]. Although  ERBB3  contains an impaired kinase domain, it is capable of ligand binding and preferentially hetrodimerizes with  ERBB2  to potently activate cellular signaling pathways [ 43 ]. Thus the  ERBB3  mutations described here are predicted to cooperate with  ERBB2  to promote ligand-independent oncogenic transformation, as functionally demonstrated for other kinase and extracellular mutations in this gene [ 41 ]. Of note, the  ERBB3  mutant carcinoma was also HER2+. We also identified a single somatic extracellular domain mutation in another ErbB receptor,  ERBB4  (p.Glu57Asp).\nWe also identified somatic mutations in epigenetic regulatory genes, including the chromatin-remodeling factors  ARID1A  (1/5 benign MOTs and 1/11 carcinomas; a predicted significantly mutated gene) and  ARID2  (1/11 carcinomas) (Table  2 , Fig.  2 ). Both genes are recognized suppressors of tumorigenesis in multiple cancer types [ 44 ,  45 ]. Consistent with this, the two  ARID1A  mutations result in protein truncation (p.Gln1894Profs*7 and p.Arg2116Thrfs*33). A further missense mutation was found in the Polycomb-group protein member  ASXL1 .\nIn addition to  ELF3  and  KLF5 , other genes implicated in the control of gene expression were collectively mutated in multiple samples. Three transcriptional co-regulatory proteins contained somatic mutations, including the consensus driver gene  BCL-6 corepressor  ( BCOR ; 2/11 carcinomas including splice donor and missense mutations) [ 45 ], and proposed pan-cancer drivers  NCOR2  (inframe indel in a benign tumor) and  ARHGAP35  (1/11 carcinomas) [ 46 ,  47 ]. Other genes involved in transcription also featured, such as a single mutation in  TAF1  that forms the large subunit of the transcription factor II D complex and facilitates the initiation of transcription by RNA polymerase II, and a missense mutation at the serine 34 hotspot of pre-mRNA splicing factor  U2AF1 , which has been shown to alter the cancer transcriptome [ 48 ]. The  GATA3  transcription factor was also mutated in one carcinoma.\nOne other important group of genes mutated in MOTs included those associated with ubiquitin-mediated protein degradation. As well as frequent deleterious mutations in the E3 ubiquitin ligase  RNF43  [ 8 ], the consensus cancer gene and tumor suppressor  FBXW7  is noteworthy [ 45 ], encoding for the substrate recognition component of SCF (complex of SKP1, CUL1 and F-box protein)-type ubiquitin ligases. Recurrent heterozygous mutations (1/8 borderline tumors and 1/11 carcinomas) (Table  2 , Fig.  2 ) are predicted to result in proteins with impaired (p.Asp560Asn) or absent (p.Arg278*) substrate binding capability that dominantly interfere with wildtype protein through the intact dimerization domain. Interestingly, the mutant borderline tumor also harbored bi-allelic mutations in another SCF complex gene  CUL1 . We also identified a missense mutation in the E3 ubiquitin-protein ligase and cancer gene  UBR5  [ 46 ].\nOther genes identified based on significance prediction and mutated in 2/24 MOTs by exome sequencing included leucine-rich repeat kinase 2 ( LRRK2 ), the ribosomal gene transcription termination factor  TTF1 , and  LPHN3 , which encodes a member of the latrophilin subfamily of G protein-coupled receptors (Table  2 , Fig.  2 ).  DCLK1 , a newly identified marker of transformed stem cells in the gut [ 49 ], was also mutated in 8.3 % of cases (Table  2 , Fig.  2 ), and is a recurrent target for mutation in neoplasms of the stomach [ 25 ], appendix [ 37 ] and skin [ 50 ]. Clonal heterogeneity may be a feature of  DCLK1 , as we and others [ 25 ,  50 ] observed mutations at low allelic fractions. Validation in a larger cohort of samples is needed to interpret the role of these genes in mucinous ovarian tumorigenesis.\n\nLittle is known about the genomics of mucinous ovarian carcinoma beyond the known cancer driver genes. Here, through mutation analysis, we provide insight into the somatically altered genes of MOTs, identifying many candidates not previously implicated in this disease, including a higher than expected proportion of carcinomas with  TP53  and  BRAF  mutations, as well as the prevalent RAS pathway mutations and loss of p16. Therapies for this relatively rare entity have focused on general chemotherapeutics currently used in ovarian cancer. These therapies show limited success in treating advanced mucinous disease and novel targeted therapies would be beneficial, especially for high-grade carcinoma.\nUsing exome sequencing we could resolve driver mutations in four of the six MOTs without a  KRAS  or  BRAF  oncogenic mutation. Two tumors were likely driven by alternative mechanisms for constitutive RAS signaling ( RRAS2  mutation and HER2 amplification) with both also harboring cooperating events in  TP53  and  CDKN2A . Two further tumors may be explained by a truncation in  ARID1A , and  ELF3  mutation plus homozygous  CDKN2A  loss, leaving only a benign and a borderline MOT unexplained. Given this, and the fact that a significant proportion of mutations in candidate drivers were identified among the carcinoma cohort, it is plausible that these genes represent cooperative mechanisms contributing to tumor progression rather than novel initiating events; the diversity of biological processes and pathways they involve hints at a high level of molecular heterogeneity in this contribution. This study provides a basis for understanding the diverse pathways targeted by somatic mutation in mucinous tumors of the ovary, although further functional work is required to elucidate the role of novel, less commonly affected genes with conflicting roles in the literature, such as  ELF3  and  KLF5 .\nIt is clear from this and other studies that the genes underlying MOTs are markedly different from other ovarian cancer subtypes. Genetic changes in the RAS/RAF pathway and concurrent loss of cell cycle regulation through aberrant p16 define MOTs. Some of the mutated genes we have observed have been seen in other tumor types, including in genes more commonly associated with tumors of the gastrointestinal tract, pancreas and endometrium, such as  RNF43 ,  ELF3 ,  ARID1A  and  GNAS . Comparing MOTs to mucinous-type tumors from other organ sites reveals some genetic similarities, but also some striking differences (Additional file  2 : Figure S3). Like MOTs, colorectal mucinous carcinomas are the only group in which frequent  KRAS  and  BRAF  mutations are found; mutations in both genes are absent in breast and rare gastric mucinous carcinomas, and appendiceal mucinous tumors are  BRAF  wild type. Likewise, pancreatic carcinomas and their mucinous neoplastic precursors appear not to be driven by oncogenic  BRAF , but instead are the only group apart from MOTs to harbor mutant  CDKN2A . We also identified genes novel to cancer that may reflect rarely targeted genes unique to the mucinous ovarian milieu. The initiating cell type of mucinous tumors presenting on the ovary remains to be determined; the heterogeneity of the mutations observed here as well as the mutational spectrum suggests that the ovarian surface epithelium is unlikely to be the only source.","source_license":"CC-BY-4.0","license_restricted":false}