{"paper_id":"f9558bb9-f07f-4228-a02d-9a99f6419f88","body_text":"Modern Pathology (2020) 33:2534 –2543\nhttps://doi.org/10.1038/s41379-020-0611-3\nARTICLE\nComprehensive genomic pro filing reveals ubiquitous KRAS\nmutations and frequent PIK3CA mutations in ovarian seromucinous\nborderline tumor\nRen-Chin Wu 1 ●\nShu-Jen Chen 2 ●\nHua-Chien Chen 2 ●\nKien Thiam Tan 2 ●\nShih-Ming Jung 1 ●\nChiao-Yun Lin 3,4 ●\nAn-Shine Chao 3,5 ●\nKuan-Gen Huang 3,4 ●\nHung-Hsueh Chou 3,4 ●\nTing-Chang Chang 3,4 ●\nAngel Chao 3,4 ●\nChyong-Huey Lai 3,4\nReceived: 3 April 2020 / Revised: 16 June 2020 / Accepted: 16 June 2020 / Published online: 2 July 2020\n© The Author(s), under exclusive licence to United States & Canadian Academy of Pathology 2020\nAbstract\nThe molecular underpinnings of seromucinous borderline tumor (SMBT) – an uncommon ovarian epithelial neoplasm\ncharacterized by association with endometriosis, frequent bilateral ovarian involvement, and occasional progression to\ninvasive carcinoma – remain poorly understood. Here, we sought to comprehensively characterize the mutational landscape\nof SMBT and elucidate the clonal relationship between bilateral ovarian SMBTs. We also compared the mutational pro files\nbetween SMBTs and concurrent invasive carcinomas. Formalin- fixed, paraf fin-embedded tissue specimens were retrieved\nfrom 28 patients diagnosed with SMBT. Massively parallel sequencing of 409 cancer-related genes was conducted to\nidentify somatic mutations in 33 SMBT samples and four concurrent invasive carcinoma specimens. TERT promoter\nmutations were assessed by Sanger sequencing, whereas immunohistochemistry was used as a surrogate tool for detecting\ndeletions or epigenetic silencing of relevant tumor suppressor genes. Twenty-six (92.9%) of the 28 patients were diagnosed\nwith stage I SMBTs. Seven (25%) cases showed bilateral ovarian involvement and 13 (46%) had concomitant endometriosis.\nConcurrent ovarian carcinomas were identi fied in three patients, whereas one case had a synchronous endometrial\ncarcinoma. Somatic mutations in the KRAS, PIK3CA, and ARID1A genes were identi fied in 100, 60.7, and 14.3% of SMBT\nsamples, respectively. In contrast, TERT promoter mutations and DNA mismatch repair de ficiencies were absent.\nSequencing of paired specimens from patients with bilateral SMBT revealed the presence of at least two shared somatic\nmutations, suggestive of a clonal relationship. Similarly, we identi fied shared somatic mutations between SMBT samples\nand concurrent ovarian carcinoma specimens. Taken together, these findings demonstrated a distinct mutational landscape of\nSMBT in which (1) KRAS is invariably mutated, (2) PIK3CA is frequently mutated, and (3) TERT promoter mutations and\nDNA mismatch repair de ficiencies are absent. Our findings represent the first extensive characterization of this rare ovarian\nneoplasm, with potential implications for disease classi fication and molecular diagnostics.\nIntroduction\nSeromucinous borderline tumor (SMBT) – also known as\natypical proliferative seromucinous tumor and endocervical-\ntype (müllerian) mucinous borderline tumor – is an\n* Angel Chao\ndrangiechao@gmail.com\n* Chyong-Huey Lai\nlaich46@cgmh.org.tw\n1 Department of Pathology, Chang Gung Memorial Hospital and\nChang Gung University, Linkou Medical Center,\nTaoyuan, Taiwan\n2 ACT Genomics, Co. Ltd., Taipei, Taiwan\n3 Department of Obstetrics and Gynecology, Chang Gung Memorial\nHospital and Chang Gung University, Linkou Medical Center,\nTaoyuan, Taiwan\n4 Gynecologic Cancer Research Center, Chang Gung Memorial\nHospital, Taoyuan, Taiwan\n5 New Taipei City Municipal Tucheng Hospital, New Taipei City,\nTaiwan\nSupplementary information The online version of this article ( https://\ndoi.org/10.1038/s41379-020-0611-3) contains supplementary\nmaterial, which is available to authorized users.\n1234567890();,:\n1234567890();,:\n\nuncommon and still incompletely characterized ovarian\nepithelial neoplasm [ 1–3]. Although SMBT was formerly\nconsidered as a subtype of mucinous borderline tumor, the\nclinical presentation of SMBT more closely resembles that\nof serous borderline tumor – with frequent bilateral invol-\nvement of the ovary and sporadic extraovarian implantation\n[3–7]. Histologically, SMBT shows the hierarchical papil-\nlary architecture typical of serous borderline tumor. How-\never, its papillae are generally lined by endocervical-like\nmucinous epithelial cells and frequently admixed with a\nvariety of müllerian epithelial cells – including\nserous (ciliated) cells, endometrioid cells, clear cells,\nsquamous cells, and “indifferent cells ” with abundant\neosinophilic cytoplasm [ 3–6]. Approximately one third of\nSMBTs are associated with endometriosis, which is\nseldom found in patients with serous or mucinous border-\nline tumors [ 3].\nAlthough SMBT generally portends an excellent prog-\nnosis (with only a few cases of recurrence and a single death\nreported in the literature) [ 8], the neoplasm may display\nunfavorable histological features − including micro-\npapillary growth pattern, intraepithelial carcinoma, and\npresence of microinvasion [ 3–6]. Furthermore, SMBT may\nprogress toward a form of invasive carcinoma termed ser-\nomucinous carcinoma (SMC) by the 2014 World Health\nOrganization (WHO) Classi fication of Tumors of Female\nReproductive Organs [ 9]. Currently, SMC cannot be con-\nsidered a histologically well-de fined entity but rather shares\nsignificant morphological and immunophenotypical over-\nlaps with other types of ovarian tumors – particularly\nendometrioid carcinoma and low-grade serous carcinoma\nwith mucinous differentiation [ 10, 11]. Owing to its low\nmorphologic reproducibility and lack of de fining genetic\ncharacteristics, some pathologists have even proposed to\ndiscontinue the term SMC [ 10].\nThe molecular underpinnings of SMBT remains poorly\nunderstood as well. Mutational analysis of SMBT has only\nbeen performed in one study, which solely focused on\nKRAS and PTEN mutations in 16 samples [ 12]. KRAS\nmutations were found to be common (69% of cases),\nwhereas no PTEN mutations were detected – resulting in a\ngenetic pro file similar to that of mucinous borderline tumor\n(of gastrointestinal type) [ 12]. Another report demonstrated\nthe loss of immunohistochemical expression of ARID1A (a\nsurrogate for ARID1A mutations) in 33% of SMBT samples,\ni.e., a frequency in line with that of other endometriosis-\nrelated ovarian neoplasms (clear cell carcinoma and endo-\nmetrioid carcinoma) [ 13]. Here, we used massively parallel\nsequencing to comprehensively decipher the mutational\nlandscape of SMBT – with a special focus on the clonal\nrelationships between tumors showing a bilateral presenta-\ntion. In an effort to shed more light on the controversial\nissue of SMC, we also investigated the genetic\ncharacteristics of invasive carcinomas that arose in asso-\nciation with SMBTs.\nMaterials and methods\nPatient identi fication and retrieval of tissue\nspecimens\nWe searched our departmental surgical pathology archives\nand identi fied 44 patients with ovarian tumors diagnosed\nas either seromucinous borderline (or borderline ser-\nomucinous) tumor ( n = 37) or endocervical-type (or -like)\nmucinous borderline tumor ( n = 7) between January 1,\n2005 and December 31, 2016. Two pathologists (R.C.W.\nand S.M.J.) independently reviewed all hematoxylin and\neosin-stained slides to ide ntify SMBT specimens. Seven\ncases were excluded because the tumor histology is not\ntypical for SMBTs. Additiona l 9 patients were excluded\nbecause the tumor sizes were deemed too small for further\nmolecular analysis (atypical proliferative part <5 mm).\nThe final study cohort consisted of 28 patients with a\nconfirmed pathological diagnosis of SMBT (21 unilateral\nand seven bilateral). Concurrent invasive ovarian carci-\nnoma adjacent to SMBT was evident in three patients,\nwhereas one case had a synchr onous uterine endometrioid\ncarcinoma. For the purpose of the study, the following\nformalin- fixed, paraf fin-embedded (FFPE) tissue speci-\nmens were retrieved: SMBT ( n = 33, including five cases\nwith bilateral disease), SM BT-concurrent carcinoma ( n =\n4), and matched benign control tissue from lymph nodes,\nfallopian tubes, or uterus ( n = 28). Clinical characteristics\nincluded age at diagnosis, disease stage, type of surgery,\nand survival figures. Tissue specimens were drawn from\nthe tissue bank of the Chang Gung Memorial Hospital\n(Taoyuan, Taiwan) after ethical approval was granted by\nthe Institutional Review B oard of Chang Gung Memorial\nHospital (approval number: 201701220B0). Owing to the\nretrospective nature of the study, the need for informed\nconsent was waived.\nSample processing and DNA extraction\nA pathologist (R.C.W.) performed a careful selection of\nrepresentative FFPE blocks and identi fied areas of SMBT or\ninvasive carcinomas suitable for macrodissection. A thor-\nough manual dissection of different tumor components was\nconducted on 10- μM-thick tissue sections to reduce con-\ntamination and – in case of invasive carcinoma samples – to\nrule out the presence of non-invasive tissue. Genomic DNA\nfor mutation analysis was extracted from FFPE samples\nusing a commercially available kit (Qiagen Inc., Valencia,\nCA, USA) as previously described [ 14, 15].\nComprehensive genomic pro filing reveals ubiquitous KRAS mutations and frequent PIK3CA mutations in. . . 2535\n\nMassively parallel sequencing and data analysis\nPaired tumor and normal samples were subjected to mas-\nsively parallel sequencing targeting the coding regions of\n409 cancer-related genes. The sequencing procedures and\nthe approach used for data analysis have been previously\ndescribed in detail [ 14, 15]. In brief, genomic DNA from\neach sample (80 ng) was ampli fied by polymerase chain\nreaction (PCR) to enrich the coding exons of targeted genes.\nTo this aim, AmpliSeq Comprehensive Cancer Panel primer\npools (Thermo Fisher Scienti fic, Waltham, MA, USA) were\nused. Amplicons were subsequently ligated with barcoded\nadaptors, conjugated with sequencing beads, and enriched\nusing Ion Chef (Thermo Fisher Scienti fic) according to the\nIon Torrent protocol. Sequencing was performed on an Ion\nProton sequencer using the Ion PI chip (Thermo Fisher\nScientific). Raw data from the sequencer were mapped to\nthe hg19 reference genome using the Ion Torrent Suite\n(v. 4.2). Single nucleotide variants and short insertion/\ndeletions were identi fied with the Torrent Variant Caller\nplug-in (v. 4.2). All variants were annotated using Variant\nEffect Predictor (VEP, release 78) and filtered out when\ntheir frequency was <5% or in presence of <50 reads.\nVariants not identi fied in matched normal samples were\nconsidered as somatic mutations. Further annotation was\nperformed using COSMIC (v. 70), dbSNP (138), and 1000\nGenomes (phase 1).\nMutation analysis of the TERT promoter\nThe promoter region of the TERT gene – which is known to\ncontain two mutation hotspots (chr5: 1,295,228 and\n1,295,250; hg19) – was ampli fied by PCR using the fol-\nlowing primers: 5 ʼ-M13-CAGCGCTGCCTGAAACTC-3ʼ\nand 5 ʼ-GTCCTGCCCCTTCACCTT-3ʼ, where M13 indi-\ncates a universal sequencing primer (5 ʼ-GTAAAAC\nGACGGCCAGT-3ʼ). PCR conditions were as follows:\n95 °C for 5 min, followed by 45 cycles at 98 °C for 20 s,\n60 °C for 15 s, and 72 °C for 1 min, followed by a final\nextension at 72 °C for 5 min. Ampli fied PCR products were\npurified and subjected to Sanger sequencing.\nImmunohistochemistry\nImmunohistochemical staining of 3-µm-thick paraf fin sec-\ntions was performed using the following antibodies:\nARID1A (1:200 dilution; clone HPA005456, Milli-\nporeSigma, St. Louis, MO, USA), ER (1:200; clone 6F11,\nLeica Biosystems), MLH1 (1:50 dilution; clone GM011,\nGenemed Biotechnologies, Torrance, CA, USA), Napsin A\n(1:200 dilution; clone IP64, Leica Biosystems), p53 (1:100\ndilution; clone DO7, Leica Biosystems, Buffalo Grove, IL,\nUSA), PAX8 (1:50 dilution; clone BC12, Biocare, Pacheco,\nCA, USA), PR (1:400 dilution; clone 16, Leica Biosys-\ntems), PTEN (1:100 dilution; clone 138G6, Cell Signaling,\nDanvers, MA, USA), and WT1 (1:200 dilution; clone 6F-\nH2, Quartett, Berlin, Germany). Immunostaining was per-\nformed on a BOND-MAX automated stainer (Leica Bio-\nsystems). Heat-induced epitope retrieval was performed at\n100 °C using citrate-based pH 6.0 buffer (BOND Epitope\nRetrieval Solution 1, Leica Biosystem) for Napsin A, and\nEDTA-based pH 9.0 buffer (BOND Epitope Retrieval\nSolution 2, Leica Biosystems) for the other antibodies.\nImmunoreactivity was assessed with a BOND Polymer\nRefine Detection system (Leica Biosystems). For ER, PR,\nNapsin A, and PAX8, reactions were interpreted as positive\nif at least 5% of tumor cells showed expression.\nResults\nPatient characteristics\nThe clinical characteristics of the 28 patients with SMBT\n(unilateral, n = 21; bilateral, n = 7) are summarized in\nTable 1. The median age at diagnosis was 37 years (range,\n25–58 years). In general, patients presented with early-stage\ndisease with 26 (92.9%) diagnosed at stage I and two at\nstage II, with a median tumor size of 7.7 cm (range,\n2.7–15.2 cm). The median length of follow-up was\n38.8 months (range, 14.6 –240.6 months). Disease recur-\nrence was observed in one case only (S20). The patient\ninitially underwent surgical removal of an SMBT (stage II)\ninvolving the left ovary and the pelvic peritoneum. Twenty-\nseven months thereafter, a right ovarian SMBT was diag-\nnosed and excised. A recurrence of SMBT at the right ovary\nwas observed at 127 months of follow-up. The patient was\nsuccessfully salvaged with hysterectomy and right salpingo-\noophorectomy. At the time of last follow-up, all participants\nwere alive without disease.\nHistopathologic characteristics of seromucinous\nborderline tumors\nAll SMBTs were characterized by the presence of hier-\narchical papillary structures lined by endocervical-like\nmucinous epithelial cells and cilia-bearing serous epithe-\nlial cells (Fig. 1a, b). Moreover, variable amounts of\nendometrioid cells, squamous cells, piling-up clear cells,\nand “indifferent cells ” with ample eosinophilic cytoplasm\nwere observed (Fig. 1b–d). Nuclear atypia in SMBT cells\nwas generally mild-to-moderate. All SMBTs showed, at\nleast focally, prominent intraepithelial and mesenchymal\nneutrophilic infiltration (Fig. 1c, d). Five cases (17.9%) had\nevidence of focal intraepithelial carcinoma characterized by\nexuberant cribriform proliferation over papillary surfaces or\n2536 R.-C. Wu et al.\n\ncystic linings (Fig. 1e–f). There was no evidence of\nmicropapillary growth pattern or microinvasion in any of\nthe study specimen. Three cases had foci of invasive growth\nadjacent to SMBT (all >5 mm in their greatest dimension)\nand their histological and genetic features will be subse-\nquently described in detail. There were two cases in whom\nSMBT was associated with mature cystic teratoma ( n = 1)\nand synchronous uterine endometrioid carcinoma ( n = 1). A\ntotal of 13 (46%) patients had concomitant endometriosis\n(Fig. 2a). All endometriotic lesions were cystic involving\novary in 13 patients.\nMutational landscape of seromucinous borderline\ntumors\nMassively parallel sequencing identi fied a median of\nthree somatic mutations (range, 1 –1 0 )i ne a c hs e q u e n c e d\nSMBT specimen. Six genes were found mutated in at least\ntwo cases (Fig. 2a; Supplementary Fig. 1). Strikingly,\nsomatic KRAS mutations were invariably identi fied in all\nSMBT samples – all of them being hotspot mutations\ninvolving codons 12 or 13 (p.G12A, p.G12C, p.G12D,\np.G12V, and p.G13D). Somatic mutations of the PIK3CA\nTable 1 Characteristics of patients with seromucinous borderline tumors.\nPatient ID Age\n(years)\nDisease stage Surgery type Laterality Tumor\nsize (mm)\nFollow-up\n(months)\nSample ID\nS01 38 IB LAVH +BSO+Om+Ap B L50/R19 14.6 S01L\nS02a 40 IB RSO +Lenu B L27/R99 17.4 S02L/R\nS03 43 IA LSO +Renu L 60 18.2 S03L\nS04 32 IC RSO +Lenu (LS) R 65 20.3 S04R\nS05 39 IC RSO +Lenu R 80 23.6 S05R\nS06 40 IA ATH +BSO+Om+Ap+BPLN L 78 24.9 S06L/EM b\nS07 25 IC Lenu (LS) L 54 26.6 S07L\nS08 47 IC RSO R 67 24.6 S08R/R_ca c\nS09 29 IC RSO (LS) R 45 29.1 S09R\nS10 32 IC Renu (LS) R 27 34 S10R\nS11 30 IC LSO +Renu+Om+Ap+LPLN L 69 19.5 S11L\nS12 26 IC LSO (LS) L 125 44.7 S12L\nS13 31 IC Renu +Lenu+BPLN (LS) R 112 26.6 S13R\nS14 49 IC ATH +BSO+Om R 76 43.6 S14R\nS15 33 IC RSO +RPLN+Om+Ap+Bx R 152 25.4 S15R\nS16\na 32 IB LSO +Renu B L92/R85 77.8 S16L/R\nS19 30 IB Renu +Lenu B L65/R105 73.9 S19R\nS20 38 II LSO +Om+LPLN ->Renu ->\nRSO+ATH\nL ->R\n(recurrent)\n100 240.6 S20R\nS21a 51 IB ATH +BSO+BPLN+Ap+Om B L35/R140 97.4 S21L/R/R_ca d\nS23 41 IC BSO (LS) B L70/R70 102.4 S23R\nS24 35 IC RSO +RPLN+Om+P Bx R 70 115.5 S24R\nS26 47 IC ATH +BSO R 105 128.6 S26R\nS27 40 IC RSO +Lenu+RPLN+Om+Ap R 115 17.2 S27R\nS28a 29 IB LSO +Renu+BPLN+Om+Ap B L115/R118 58.3 S28L/R\nS29 31 IC ATH +RSO+Lenu+Om+Ap R 109 120.5 S29R/R_ca d\nS31 49 IC ATH +BSO L 90 133.7 S31L\nS32a 58 IB LSO +Renu B L51/R29 53.8 S32L/R\nS33 30 II LSO +Renu+CDS Bx L L85 61.9 S33L\nATH abdominal total hysterectomy, LAVH laparoscopically-assisted vaginal hysterectomy, BSO bilateral salpingo-oophorectomy, BPLN bilateral\npelvic lymphadenectomy, RSO right salpingo-oophorectomy, LSO left salpingo-oophorectomy, LS laparoscopy, Ap appendectomy, Om\nomentectomy, Lenu left enucleation, Renu right enucleation, LPLN left pelvic lymphadenectomy, CDS cul-de-sac, P peritoneum, Bx biopsy.\naBilateral seromucinous tumors were subjected to sequencing.\nbConcurrent uterine endometrioid carcinoma.\ncConcurrent ovarian clear cell carcinoma.\ndConcurrent ovarian seromucinous carcinoma.\nComprehensive genomic pro filing reveals ubiquitous KRAS mutations and frequent PIK3CA mutations in. . . 2537\n\nand PTEN genes were identi fied in 60.7% (17/28) and\n3.6% (1/28) of the SMBT specimens, respectively, in a\nmutually exclusive fashion. Four (14.3%) patients har-\nbored ARID1A truncating mutations, whereas somatic\nmutations in the KMT2C, TET2,a n d ZNF521 genes were\nidentified in two (7.1%) cases. No mutations in genes\ncoding for DNA mismatch repair proteins (i.e., MLH1,\nMSH2, MSH6, PMS1,a n d PMS2) were identi fied. Simi-\nlarly, Sanger sequencing did not identify TERT promoter\nmutations. A complete list of somatic mutations detected\nFig. 1 Representative\nphotomicrographs of\nseromucinous borderline\ntumors (SMBTs). a Presence of\nhierarchical papillary structures.\nb Lined by an admixture of\nmucinous cells, cilia-bearing\ncells. c Endometrioid cells,\npiling-up clear cells. d Piling up\nsquamous cells with mild-to\nmoderate nuclear atypia, ( c and\nd) prominent neutrophilic\ninfiltration was evident. e An\nSMBT specimen with a region\nof intraepithelial carcinoma\n(lower portion of the figure). f\nIntraepithelial carcinoma\ncharacterized by an exuberant\ncribriform proliferation over\npapillary surfaces and cystic\nlinings.\nFig. 2 Oncoplots summarizing nonsynonymous somatic mutations\nidentified in seromucinous borderline tumor (SMBT) specimens. a\nList of the six most commonly mutated genes (in decreasing order\nfrom the top to the bottom). The five cases below the orange bar had\nbilateral disease (both lesions subjected to sequencing). b A clonal\nrelationship between bilateral SMBTs was evident when the somatic\nmutations detected in paired samples were compared. The concomitant\npresence of endometriosis (EMOsis) and intraepithelial carcinoma\n(IEC) is reported in the first two rows.\n2538 R.-C. Wu et al.\n\nFig. 3 Representative\nimmunohistochemical staining\nof MLH1, p53, PTEN, and\nARID1A in seromucinous\nborderline tumor (SMBT)\nspecimens (20 × objective\nlens). a An intact MLH1 nuclear\nexpression was evident in all\nsamples. b A heterogeneous,\n“wildtype” p53 staining pattern\nwas detected in all specimens.\nc Loss of PTEN expression in an\nSMBT sample (S26R). d Loss of\nARID1A nuclear expression in\nan SMBT sample (S31L).\nFig. 4 Histological and genetic\ncharacteristics of carcinomas\nthat arose concurrently with\nseromucinous borderline\ntumors (SMBTs). a A case\n(S29) of SMBT (left upper part\nof the image) with an adjacent\ninvasive ovarian carcinoma\n(right lower part, 4× objective\nlens). b The invasive ovarian\ncarcinoma diagnosed in case\nS29 – a seromucinous carcinoma\n(SMC) – consisted of con fluent\nglandular structures lined by\nendocervical-like mucinous,\nendometrioid, and eosinophilic\nindifferent cells (20× objective\nlens). c The SMC diagnosed in\ncase S21 was characterized by\nthe presence of glandular\nstructures lined by mucinous,\nendometrioid, and squamoid\ncells (20× objective lens). d The\nSMC diagnosed in case\nS21 showed focal neoplastic\nglands lined by cells with clear\ncytoplasm (20× objective lens).\ne A case (S08) of ovarian clear\ncell carcinoma that arose\nadjacent to SMBT; note the\npresence of tubulopapillary\nstructures lined by clear cells\n(20× objective lens). f Oncoplot\ncomparing the mutational\nlandscapes of SMBT and\nconcurrent ovarian carcinomas.\nComprehensive genomic pro filing reveals ubiquitous KRAS mutations and frequent PIK3CA mutations in. . . 2539\n\nin SMBT specimens is provided in Supplementary\nTable 1.\nClonal relationship between bilateral seromucinous\nborderline tumors\nWe subsequently focused on cases with bilateral SMBT\n(n = 7). Of them, we were able to extract suf ficient amounts\nof high-quality DNA from paired samples derived from five\npatients. Targeted massively parallel sequencing revealed\nthat all paired specimens shared at least two somatic\nmutations (range: 2 –4), suggesting that bilateral SMBTs\nwere clonally related to each other (Fig. 2b). All of the five\npaired samples subjected to sequencing had an intact cap-\nsule without tumor cells over the ovarian surface – a finding\nthat argued against the metastatic nature of the contralateral\nlesion.\nImmunohistochemical analysis\nAll SMBTs were positive for PAX8 and negative for WT1.\nER was expressed in all SMBTs and PR in most (91%)\nSMBTs. Napsin A was focally expressed (<25%) in two\nSMBT samples (S02R, S16R). The aforementioned results\nwere in line with previous studies [ 16, 17], supporting the\nhistological diagnosis of SMBT in our study cohort.\nWe analyzed the immunohistochemical expression of\nARID1A, MLH1, p53, and PTEN in an effort to identify\npotential epigenetic silencing or large genomic deletions\nundetectable by targeted sequencing. All of the SMBT\nspecimens were characterized by diffuse nuclear expression\nof MLH1 (Fig. 3a). These results indicate that epigenetic\nsilencing of MLH1 – which is commonly encountered in\nendometrioid carcinoma – is a rare event in SMBT. All of\nthe samples showed heterogeneous p53 expression, sug-\ngesting that TP53 genetic aberrations are invariably absent\nin SMBTs (Fig. 3b). Loss of PTEN expression was iden-\ntified in a single specimen (sample S26R) known to harbor a\nsomatic PTEN mutation (Fig. 3c). ARID1A expression was\nundetectable in three SMBT samples (S01L, S26R, and\nS31L), all of them carrying ARID1A mutations (Fig. 3d).\nNotably, the associated ipsilateral endometriosis also lost\nARID1A expression (S01L and S26R). Taken together, the\nresults of immunohistochemistry did not detect molecular\naberrations other than those already identi fied by targeted\nsequencing.\nMutational analysis of concurrent gynecologic\ncancers in patients with seromucinous borderline\ntumor\nOf the 28 study patients, three (S08, S21, and S29) had a\nconcurrent invasive ovarian carcinoma adjacent to the\nSMBT (Fig. 4a). A fourth case (S06) was diagnosed with\nsynchronous uterine endometrioid carcinoma. The invasive\nlesions identi fied in cases S21 and S29 were classi fied as\nSMC. In case S21, we observed an admixture of\nendocervical-like mucinous, endometrioid, and clear cells\narranged predominantly in a con fluent glandular pattern and\nfocally in a solid pattern. In case S29, variable amounts of\nendocervical-like mucinous, endometrioid, and “indifferent\ncells” with abundant eosinophilic cytoplasm were structured\nin a con fluent glandular pattern (Fig. 4b-d). The immuno-\nprofiles of both SMCs were identical to those of the cor-\nresponding SMBTs. The invasive component of case\nS08 showed the typical histological findings of clear cell\ncarcinoma – consisting of tumor cells with clear cytoplasm\ngrowing in either a tubulopapillary or solid pattern (Fig. 4e).\nImmunohistochemically, the clear cell carcinoma was\npositive for Napsin A and negative for ER and PR, whereas\nthe adjacent SMBT was negative for Napsin A and positive\nfor hormonal receptors.\nMassively parallel sequencing revealed that all of the\nthree concurrent invasive ovarian carcinomas shared certain\nsomatic mutations with concomitant SMBTs – supporting\nthe existence of a clonal relationship (Fig. 4f). The fol-\nlowing three shared mutations were identi fied in patient\nS08: KRAS (c.35G>A), PIK3CA (c.1633G>A), and\nZNF521 (c.2836C>T). Five shared mutations were detected\nin patient S21, as follows: KRAS (c.35G>T), PIK3CA\n(c.3140A>G), NCOA2 (c.1991C>A), NTRK3 (c.446C>T),\nand USP9X (c.4548_4551delCAAA). A single shared\nmutation – KRAS (c.38G>A) – was identi fied in patient\nS29. The SMC identi fied in patient S21 also carried a\nmutation in NTRK1 (c.1486_1487insC) – which was absent\nin the adjacent SMBT and may theoretically be involved in\nthe progression from SMBT to SMC (Fig. 4f). In contrast,\nthe synchronous uterine endometrioid carcinoma diagnosed\nin patient S06 did not share any somatic mutation with its\ncoexisting SMBT (Supplementary Table 2).\nDiscussion\nThis study is the first comprehensive attempt to shed more\nlight on the molecular underpinnings of SMBT through the\napplication of next-generation sequencing on carefully\ndissected tumor specimens. Our main results can be sum-\nmarized as follows. First, SMBT was found to have unique\nmolecular features that set it apart from both other border-\nline tumors of the ovary and endometriosis-associated\nneoplasms. Speci fically, the SMBT signature consisted of\nfrequent somatic mutations in the KRAS (100%), PIK3CA\n(60.7%), and ARID1A (14.3%) genes, with TERT promoter\nmutations and DNA mismatch repair de ficiencies being\nconsistently absent. Second, we show that bilateral SMBTs\n2540 R.-C. Wu et al.\n\nare frequent (25%) and clonally related to each other – as\nattested by the presence of shared somatic mutations. Third,\na similar clonal relationship was identi fied between SMBT\nand concurrent ovarian carcinomas – including SMC and\nclear cell carcinoma. In light of these findings, the presence\nof KRAS mutations may serve as a genetic hallmark of\nSMBT – a finding that con firm and expand previous\nobservations [ 12].\nFormerly known as endocervical-type mucinous bor-\nderline tumor, SMBT was reclassi fied as a separate disease\ncategory in the latest 2014 WHO Classi fication of Tumors\nof Female Reproductive Organs [ 9]. Because the term\n“seromucinous” may cause confusion with serous and\nmucinous borderline tumors, Kurman and Shih have pre-\nviously recommended the term “mixed müllerian borderline\ntumor” and emphasized the morphological and immuno-\nhistochemical differences between SMBT and serous/\nmucinous borderline tumor [ 18]. Our study provides further\nmolecular genetic evidence that distinguishes SMBT from\nserous/mucinous borderline tumor. Differently from serous\nborderline tumor – which is characterized by mutually\nexclusive mutations in KRAS (17–39.5% of cases) and\nBRAF (23–48% of cases) [ 19] – our SMBT samples were\nfound to invariably harbor KRAS mutations, with BRAF\nbeing rarely mutated (3.6%). Notably, a very high rate of\nKRAS mutations (92.3%) has been previously reported in\nmucinous borderline tumor [ 20] – potentially indicating a\nshared molecular basis with SMBT. However, PIK3CA\nmutations – which were identi fied in 60.7% of our SMBT\nsamples – seem to occur much less frequently in MBT\n(15%) [ 20]. Moreover, we did not identify CDKN2A\nmutations – previously reported in 19.2% of MBT cases\n[20] – in our SMBT specimens.\nAlthough SMBT is frequently associated with endome-\ntriosis (46% of cases in the current study), our data indicate\nthat the mutational landscape of SMBT is distinct from that\nof other endometriosis-associated neoplasms − including\novarian endometrioid carcinoma and clear cell carcinoma\n(Table 2)[ 21–30]. Differently from ovarian endometrioid\ncarcinoma, CTNNB1 mutations, loss of PTEN expression,\nor DNA mismatch repair de ficiencies were rarely identi fied\nin our SMBT specimens [ 22, 23, 30]. In addition, SMBT\nwas distinct from ovarian clear cell carcinoma, as TERT\npromoter mutations were not found in SMBT whereas\nKRAS mutations were ubiquitous in SMBT [ 24, 25].\nNot uncommonly, patients with SMBT show bilateral\novarian involvement at presentation [ 31]. Here, we\ndemonstrated for the first time that bilateral ovarian SMBTs\nwere clonally related to each other. Two potential expla-\nnations for this clonal relationship could be offered,\nincluding: (1) metastatic spread of a primary SMBT to the\ncontralateral ovary, or (2) independent onset of contralateral\nSMBT from clonal endometriotic lesions affecting both\novaries. Notably, a similar clonal relationship has been\npreviously reported for bilateral ovarian serous borderline\ntumor, which is likely attributable to contralateral ovarian\nmetastasis owing to the frequent presence of ovarian surface\ninvolvement by these neoplasms [ 31]. However, all bilateral\nSMBTs identi fied in our study were characterized by\nan intact capsule and the absence of tumor cells over\nthe ovarian surface. These observations argue against a\nmetastatic origin and support the view that bilateral\nSMBTs arise independently of each other – most likely\nfrom clonally related bilateral ovarian endometriotic lesions\nharboring driver mutations in KRAS, PIK3CA, or other\ngenes [ 32, 33].\nSMC – a poorly characterized entity of ovarian epithelial\ncancer introduced in the 2014 WHO Classi fication of\nTumors of Female Reproductive Organs [ 9] − can exhibit a\nwide variety of histopathological features consisting of an\nadmixture of different cell types (including endocervical-\nlike mucinous, endometrioid, eosinophilic “indifferent”,\nhobnail, squamous, signet-ring, and clear cells) [ 11]. The\nquestion as to whether SMC should be regarded as a distinct\ncategory of ovarian cancer is still a matter of debate owing\nto its obvious morphological overlaps with low-grade ser-\nous, mucinous, and endometrioid carcinomas [ 10, 11].\nFrom the perspective of multistep carcinogenesis, it is rea-\nsonable to regard as “true” SMC those arising in association\nwith SMBT – which accounted for approximately 50% of\ncases previously reported in a large series [ 11]. SMBT-\nassociated SMCs are likely to derive from SMBTs – as\nshown by the clonal relationship between SMBTs and\nSMCs identi fied in our study. They should be possibly\nregarded as a genetically distinct group of neoplasms in\nwhich KRAS and PIK3CA are commonly mutated. How-\never, the differential diagnosis between SMCs without a\ncoexisting SMBT component and other histotypes (espe-\ncially endometrioid carcinoma with mucinous\nTable 2 Comparison of molecular aberrations among endometriosis-\nassociated ovarian neoplasms.\nGene SMBT CCC EMCA\nKRAS 100% 4.7 –7% [ 24, 27] 33.3% [ 22]\nPIK3CA 60.70% 33 –43% [ 21, 24, 27] 40.0% [ 22]\nARID1A 14.30% 46 –57% [ 26, 27] 33.0% [ 26]\nPTEN 3.6%a&b 5%b [24] 23.9% a [23]\nCTNNB1 3.60% 1.0% [ 24] 53.3% [ 22]\nTERT promoter 0% 15.9% [ 25]0 % [ 25]\ndMMR 0% a&b 6%a [28, 29] 11.3% a [30]\nSMBT seromucinous borderline tumor, CCC clear cell carcinoma,\nEMCA endometrioid carcinoma, dMMR deficient DNA mismatch\nrepair.\naDetected by immunohistochemistry.\nbBy sequencing.\nComprehensive genomic pro filing reveals ubiquitous KRAS mutations and frequent PIK3CA mutations in. . . 2541\n\ndifferentiation) is challenging at best and frequently\nimpossible [ 10]. Because KRAS is invariably mutated in\nSMBT, it can be hypothesized that the absence of KRAS\nsomatic mutations may help identify at least certain SMC\nmimickers. Unfortunately, KRAS mutations are not\nuncommon in endometrioid carcinoma with mucinous dif-\nferentiation – ultimately limiting the diagnostic utility of\nthis molecular approach [ 10, 34].\nIn our study, we identi fied one patient with SMBT and\nconcurrent ovarian clear cell carcinoma – with the two\nlesions being clonally related. To our knowledge, only\nanother similar case has been reported in the literature [ 35].\nWhile this observation seems to suggest that SMBT may act\nas a precursor to clear cell carcinoma, such lesions might as\nwell be collision tumors. Indeed, an SMBT and a clear cell\ncarcinoma within a collision tumor may appear clonally\nrelated to each other if they arise independently from the\nsame endometriotic cyst that already harbors cancer driver\nmutations.\nIn conclusion, our current data represent the first exten-\nsive characterization of SMBT in terms of histology,\nimmunohistochemistry, and molecular pathogenesis. If\nindependently con firmed, our findings may have signi ficant\nimplications for disease classi fication and molecular\ndiagnostics.\nAcknowledgements This study was supported by the Chang Gung\nMedical Foundation, Taiwan (grants CRRPG3F0041/2/3,\nCMRPG3H1151/2, and CMRPG3H0351/2/3).\nCompliance with ethical standards\nConflict of interest The authors declare that they have no con flict of\ninterest.\nPublisher’s note Springer Nature remains neutral with regard to\njurisdictional claims in published maps and institutional af filiations.\nReferences\n1. Rutgers JL, Scully RE. 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