{"paper_id":"c38748f2-166b-44be-aedd-5b732127e31c","body_text":"1 \n \nMolecular analysis suggests oligoclonality and metastasis of \nendometriosis lesions across anatomically defined subtypes. \n \nRunning title: clonality and metastasis in endometriosis \n \nTeresa H. Praetorius1,2, Anna Leonova2,3, Vivian Lac2, Janine Senz2, Basile Tessier-Cloutier2,4, \nTayyebeh M. Nazeran2, Martin Köbel5, Marcel Grube1, Bernhard Kraemer1, Paul J. Yong2,3,6, \nStefan Kommoss1*, Michael S. Anglesio2,3* \n \n \nAffiliations: \n1) Department of Women’s Health, Tübingen University Hospital, 72076 Tübingen, Germany \n2) British Columbia's Gynecological Cancer Research Program (OVCARE), University of \nBritish Columbia, Vancouver General Hospital, and BC Cancer, Vancouver, BC, Canada, \nV5Z 4E6 \n3) Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, \nCanada, V6H 3Z6 \n4) Department of Pathology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, \nNew York, NY, USA, 10065 \n5) Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, Canada, \nT2N 2T9 \n6) BC Women’s Centre for Pelvic Pain & Endometriosis, BC Women’s Hospital and Health \nCentre, Vancouver, Canada, V6H 3N1 \n \n*Co-Corresponding Authors (These authors contributed equally): \nDr. Michael S. Anglesio, PhD \nm.anglesio@ubc.ca\n; University of British Columbia, Robert HN Ho Research Centre; \n2660 Oak Street, Vancouver, BC, Canada. V6H 3Z6 \n \nProf. Dr. med. Stefan Kommoss \nstefan.kommoss@med.uni-tuebingen.de\n; Department of Women’s Health, Tübingen \nUniversity Hospital, Calwerstraße 7, 72076 Tübingen, Germany  \n \n \n \n \n \n \n \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\n  \n \n2 \n \nAbstract  \nEndometriosis symptoms are heterogeneous with controversy on whether it constitutes a single \ndisease or multiple distinct types. Our previous work found recurrent somatic cancer-driver \nalterations in endometriosis; however, these have not been found ubiquitously. A handful of \ncases spread across studies also suggest mutations might be shared (clonal) between lesions \nof the same type. As current classification systems correlate poorly with symptoms or outcomes, \nsomatic genomics may improve the current system. Here, we investigate heterogeneity of \nsomatic cancer-driver mutations within patients and across endometriosis types. \nWe examined anatomically distinct types of endometriosis (ovarian, deep infiltrating, and \nsuperficial endometriosis) in 27 individual patients all of whom had at least two types of \nendometriosis. Specimens were analyzed using high-sensitivity targeted sequencing with \northogonal validation from droplet digital PCR and mutation-surrogate immunohistochemistry. \nResults found 13/27 patients had informative somatic driver mutation in endometriosis, 9/13 had \nidentical mutations across distinct lesions. Endometriomas tended to have a higher mutational \ncomplexity, with functionally redundant driver mutations in same gene and within the same \nlesions. \nOur data are consistent with clonality across endometriosis lesions regardless of subtype. \nFurther the finding of redundancy in mutations with the same gene and lesions is also \nconsistent with endometriosis representing an oligoclonal disease with dissemination likely to \nconsist of multiple epithelial clones travelling together. This suggests the current anatomically \ndefined classification of endometriosis does not fully recognize the etiology of the disease. A \nnovel classification should take into account genomic and other molecular features. These \nfindings could further contribute to development of a more personalized endometriosis diagnosis \nand care. \n \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n3 \n \nKeywords \nEndometriosis, somatic cancer-driver mutations, heterogeneity, oligoclonality, metastasis  \n \nIntroduction  \nEndometriosis is a chronic estrogen-dependent inflammatory disease defined by the presence \nof endometrial epithelial glands and stroma outside the uterine lumen and a small but significant \nrisk of malignant transformation (1-3). It is estimated to affect up to 10% of biological females of \nreproductive age and can lead to pelvic pain, dysmenorrhea, dyspareunia, and infertility as well \nas severely affect quality of life and productivity (2, 4-6). Three major anatomically described \ntypes of endometriosis are recognized: superficial peritoneal endometriosis (EM), deep \ninfiltrating endometriosis (DIE), and ovarian endometriomas (OMA) (2, 7). Current classification \nsystems such as the revised American Society for Reproductive Medicine (rASRM) scoring \nsystem and the ENZIAN classification for DIE are useful in documenting surgical findings in a \nstandardized manner. However, they poorly correlate with the severity of symptoms and fail to \nprovide a prognostic tool concerning the treatment outcome for pain or infertility  (7-9). Likewise, \ncurrent classification and staging of endometriosis do not include any information about the \nmolecular features or microenvironment of these lesions.  \nRecent studies have shown that multiple forms of endometriosis harbor somatic cancer-driver \nalterations including recurrent activating changes in KRAS, PIK3CA, ARID1A and others (10-\n12). It appears the malignant potential for these lesions remains low, despite the presence of \nrecurrent cancer-driver mutations (13-15). However, the contribution of these alterations to the \npathobiology of endometriosis remains unclear. Somatic alterations may be useful targets for \ntherapeutic intervention or tracked to study etiology and disease dissemination. The concept \nthat endometriosis disseminates is not novel. Endometriosis frequently presents with multiple \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n4 \n \nanatomical sites affected by lesions (16, 17); recent findings confirm a high rate of coexistence \nbetween OMA, EM, and DIE where only 2.3% of study had isolated OMA (16). Along with \ncurrent etiology largely attributing an origin to endometrial tissues, all endometriosis lesions may \nwell have disseminated from a eutopic point of origin. This highlights the importance of studying \nthe mechanisms of clonal dissemination and metastasis of endometriosis (12, 17, 18). Despite \nthis, there has been little research presenting objective evidence in support of widespread clonal \ndissemination of endometriosis tissues. Finally, clinical presentation of endometriosis is \nheterogeneous and it is controversial whether endometriosis constitutes one disease entity or \nwhether independent types with different underlying pathogenesis exist (19).  \nHere we explored the potential clonal relationship of endometriosis in 27 patients with multiple \nanatomically separated lesions, each having at least two distinct types of endometriosis. Our \nobjective was assessment of clonality at the level of anatomically described endometriosis types \nto address whether mutations are frequently shared between lesions and/or between lesion \ntypes. If they are not, this would suggest each anatomically defined type represents a unique \ndisease. If they are, this would support plasticity between types and/or that our understanding of \nendometriosis types is currently insufficient.  \n \nMaterials and Methods  \nExperimental subject details \nFormalin-fixed and paraffin-embedded (FFPE) archival tissues from 27 patients from the \nTübingen University Hospital, Germany were included. Inclusion criteria were confirmation of \nhistopathological diagnosis of endometriosis, presentation of two or more types of \nendometriosis (DIE, and/or EM, and/or OMA) in distinct anatomical locations, and availability of \nlesions estimated to be of sufficient size for needle macrodissection and yield of DNA for panel \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n5 \n \nsequencing and/or droplet digital PCR, as well as sectioning for immunohistochemistry (IHC). \nPatients with history of, or co-existing, malignancies were excluded. Clinical diagnosis of \nendometriosis type was extracted from patient charts and specimens were pathology-reviewed \n(by Pathologists BTC and TMN) to ensure presence of endometriosis. Experiments were done \nat the University of British Columbia and the University of Calgary. The project was conducted in \ncompliance with the Canadian Tri-Council Policy Statement on Ethical Conduct for Research \nInvolving Humans (TCPS2, 2018), effort to obtain written informed consent was exercised for all \npatients. Specimen from non-contactable patients (lost contact/deceased) treated more than 5 \nyears before the start of the study were included under institutionally approved waiver of \nconsent (Tübingen University Hospital Research Ethics Board). All institutions approved use of \nmaterials and associated clinical data through local research ethics boards. \nSample processing and DNA extraction  \nFFPE specimens were sectioned onto glass slides at 5-8um, stained with dilute hematoxylin \nand eosin, and manually enriched for endometriosis glands and stroma by needle \nmacrodissection as described previously (11, 20). DNA was extracted using the ARCTURUS® \nPicoPure® DNA Extraction Kit (ThermoFisher Scientific, USA) and quantitated using the Qubit \n2.0 Fluorometer (ThermoFisher Scientific, USA).  \nTargeted sequencing  \nDNA (45-75ng) was sequenced using a proprietary hypersensitive cancer hotspot sequencing \npanel: FIND ITTM, version 3.4 (Canexia Health, Canada) (10, 11). This assay includes hotspots \nfrom 33 genes (11, 20) (Table S1). Mutations were considered “true”, if they were genuine \nhotspot mutations targeted by the FIND IT assay and previously reported in the Catalogue of \nSomatic Mutations in Cancer (COSMIC) (21), as well as prior observations with validation (10, \n11), including a probability score >0.8 and a variant allele frequency (VAF) >0.8% (Table S2A).  \nValidation via droplet digital PCR \n \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n6 \n \nSomatic mutations identified by targeted sequencing were orthogonally validated through \ndroplet digital polymerase-chain-reaction (ddPCR) assays or IHC (for TP53 and PTEN; see \nbelow). In addition, alterations were tested by ddPCR in all available lesions, from a given \npatient, if they were observed in any one lesion from that given patient in panel-based \nsequencing. This was done even if panel testing data on other lesions was available, and \nadditionally included any specimens that were omitted from panel testing due to low DNA yield. \nUsing previously established methods (11, 20), extracted DNA was pre-amplified for targets \nover 10 cycles then diluted before assembling the ddPCR assay. Droplets were generated using \nthe QX200 Droplet Generator (Bio-Rad Laboratories, USA), amplified by thermal cycling, and \nquantified using the QX2000 Droplet Reader (Bio-Rad Laboratories, USA). As above, \nalterations were considered “true” if ddPCR droplet counts exceeded the average of the \nnegative control specimens plus 3 times the standard deviation of negative controls the relevant \nassay. See Table S2A for full listing of results and Table S3 for primer/probe details.  \nARID1A, PTEN and p53 Immunohistochemistry \n \nIHC assays for ARID1A (22, 23) , p53 (24, 25) and PTEN (11, 20) (Table S2B, S3) were used \nas surrogates for somatic alterations following established standards for staining and scoring. In \nthe case of PTEN and p53 we considered loss by IHC (see below) as sufficient for orthogonal \nvalidation of somatic mutation found in the mutation panel (none validated; Table S2) or \ndiscovery of mutation not covered in the mutation panel. ARID1A was not in our mutation panel \nbut loss was considered as “true” for discovery of somatic mutations. \nScoring was performed as follows: ARID1A loss/mutation, if nuclear staining was absent in \nendometriosis epithelium cells and internal control (stroma) was intact (22, 23). Mutant p53 \n(p53abn) if high intensity positive staining was observed in 10 or more adjacent cells in the \nepithelial cyst wall of endometriosis while maintaining a normal type pattern in surrounding \ntissue (24, 25). PTEN loss if cytoplasmic and nuclear staining was absent in endometriosis \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n7 \n \nepithelium cells and internal control (stroma) was intact (11, 20). Slides were scored by \npathologists TMN, and/or BTC, and/or MK. \nARID1A was stained on Dako Omnis automated immunostainer (Agilent Technologies, USA) or \nVentana BenchMark Ultra autostainer (Ventana Medical Systems, USA) the ARID1A rabbit \npolyclonal antibody, HPA005456 (Sigma-Aldrich). PTEN was stained on Ventana Discovery \nUltra (Ventana Medical Systems, USA) using the rabbit monoclonal antibody, 138G6 (Cell \nSignaling, USA). p53 was stained on Dako Omnis (Agilent Technologies, USA) using the p53 \nmouse monoclonal antibody DO-7 (GA61661-2; Agilent Technologies, USA).  \nStatistics \nStudent’s t-test was performed to compare the affected genes and lesion types. However, given \nour limited sample size, they remained non-significant. \n \nResults  \nWe examined 73 endometriosis lesions from 27 patients with a mean age of 34.9 years (23-45 \nyears, 60% (16/27) of patients were diagnosed with stage IV endometriosis (Table 1; Table S2; \nFigure S1). 53 lesions were subjected to panel-based sequencing with validation of selected \nalteration by ddPCR, 6 additional samples included in ddPCR validation only. We relied \nprimarily on mutation data to establish clonality (Table S4), but also included supportive \nmutation surrogate IHC data for ARID1A, PTEN, and p53. Tumor suppressors ARID1A and \nPTEN are frequently altered in endometriosis associated ovarian cancer and have been \nreported to be somatically altered at varying frequencies in endometriosis (11, 13, 23, 26, 27). \nSomatic alteration of p53 is less commonly reported in endometriosis and endometriosis \nassociated carcinomas (28-30). While our mutation panel has good coverage of the TP53 gene \nthe p53 IHC assay provided validation as well as indication of mutations not covered in the \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n8 \n \npanel assay. In total, we IHC assayed lesions for PTEN (66 interpretable), ARID1A (66 \ninterpretable) and p53 (47 interpretable). Including mutation and IHC data a total of 27/59 \n(45.8%) lesions from 13/27 (48%) cases had identifiable somatic cancer-driver mutations.  \nAmongst the panel screened lesions, nearly half of the tested cases had at least one mutation. \nPIK3CA alterations were the most common (27 hotspot mutations, affecting 12/53 lesions in 6 \ncases), followed by KRAS (16 hotspot mutations, affecting 15 lesions in 6 cases) and CTNNB1 \n(10 hotspot mutations, affecting 7/53 lesions in 4 cases; Figure 1). In contrast to finding more \nPIK3CA alterations than any others, more lesions were affected by KRAS alterations. This trend \nwas the same regardless of lesion type (Figure 1D) or if summarizing by fraction of affected \ncases (Figure 1E). Further, OMA tended to have a higher proportion of lesions affected by \nsomatic cancer-driver alterations and subsequently higher mutation load than other lesion types \n(Figure 1, Table S2). \nAlterations were less frequently observed in NRAS (6 mutations, 6/53 lesions, 4 cases), ERBB2 \n(2 mutations, 2/53 lesions, 1 case) and EGFR (2 mutations, 2/53 lesions, 2 cases). Although our \ncohort had insufficient numbers to support strong associations, we noted many lesions, \npredominantly amongst OMA, contained multiple mutations in the same genes. \nNo samples had identifiable p53-abnormal staining pattern (0/47 lesions), PTEN loss was \nobserved in 5 cases (8/66 lesions), and ARID1A loss was observed in 1 case (2/66 lesions). \nImmunohistochemical data is inconclusive with respect to clonality, and similar abnormal \npatterns were considered in support of mutation findings. Our mutation data suggested a single \nTP53 alteration but no abnormal p53 IHC pattern was observed. As IHC for p53 is accepted to \nbe a surrogate for mutation status we considered this as a false positive (case 22; Table S2A) \n(24, 25, 31). We observed no PTEN mutations in sequencing data despite evidence of loss in \nPTEN protein expression by IHC in 8/66 lesions (Table S2B), however only a few PTEN \nhotspots are covered in panel sequencing (Table S1). In case 2, with PTEN loss in multiple \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n9 \n \nlesions, other somatic (clonal) point mutations were shared between the PTEN-loss affected \nlesions (Table S2A). Case 3 showed loss of ARID1A protein consistent with loss-of function \nsomatic mutation (22) in both an EM and OMA samples (Figure 2). Unfortunately, insufficient \ntissue was available to validate any mutation in the EM sample from Case 3, however, the \nPIK3CA (p.Met1043Ile) and KRAS alterations were shared across all other lesions from this \ncase. Altogether, we observed clonality between at least two endometriotic lesions in 8 out of 13 \ninformative cases (Figure S2; Table S2; Table S4).  \nFor ease in presenting results details we have divided informative cases (n = 13) into two \ncategories (Table 1; Table S4). Simple cases had only one or two altered genes and/or one or \ntwo informative lesions. Complex cases all had alterations across lesions and at least one \nlesions with functionally redundant alterations (i.e. equivalent activating change resulting from \ndifferent nucleotide and/or amino acid substitution in the same gene). \nSimple cases\n \nSeven cases were defined as simple (Table 1). In 3 of these we identified and validated shared \nmutations across multiple lesions and types (Figure S2; Table S4). Case 6 shared mutations in \nPIK3CA between EM and DIE. The other two cases (19 and 21) had mutations in KRAS. Case \n19 shared a p.Gly12Ser mutation between an EM and a DIE. Case 21 shared a p.Gly12Asp \nbetween an OMA and three different DIE lesions. \nThe 4 remaining cases had somatic alteration that were not shared across lesions within \nindividuals, thus were not informative with respect to clonality (Figure S2). Of these, 3 cases \n(20, 22, 24), had mutations observed in only a single lesion (KRAS or EGFR) while 1 (Case 9) \nhad alterations in KRAS and PIK3CA but none shared between lesions. Case 9 did have two \n(redundant) KRAS alterations in DIE but we retained this case in our “simple” category as no \nalterations were shared between lesions.  \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n10 \n \nComplex Cases \nAll six cases in the complex category were stage IV and showed evidence of a clonal \nrelationships between lesions (Table 1, Table S4). In each case at least one lesion showed \nmultiple functionally redundant hotspot alterations (Figure S2). We observed identical somatic \nalterations shared between EM and DIE lesions in one case (case 13); between DIE and OMA \nlesions in 4 cases (cases 1, 2, 3, 8; Figure 2); and between all three types in one case (case 4; \nFigure 3). \nMultiple co-existing activating mutations (functional redundancy) were most common with \nPIK3CA: seen in 12 lesions across 5 cases. In 4/5 cases with PIK3CA mutations we observed \nco-existing alterations in CTNNB1, and in all of these at least one was shared across multiple \nsites/types within the affected patient. \nKRAS alterations were seen in 5 of 6 complex cases, only case 1 in this category showed \nmultiple redundant activating changes (p.Gly12Val in DIE, p.Gly12Ser in both DIE and OMA). \nAs PIK3CA, CTNNB1 and KRAS alteration were all relatively common we saw co-existence \nbetween all of these in 3/6 complex cases.  \nNRAS alterations were detected in 4 of 6 complex cases. In 3 cases (Cases 1, 2, 8) an NRAS \nalteration was shared between OMA and DIE. In case 4 we detected one NRAS alteration \nshared between OMA, DIE, and EM. None of the cases showed redundant alterations of NRAS \nitself but they did overlap with KRAS mutations. \nERBB2 alterations were detected in 2 of 6 complex cases. In case 3 this was shared between \nan OMA and two DIE lesions (case 3) and co-occurred with a p.Gly12Asp KRAS mutation at all \nthree sites. In case 13, the ERBB2 mutation was seen only in EM but co-occurred with an \nEGFR mutation. The same EGFR mutation was also present in DIE from case 13. \n \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n11 \n \nDiscussion  \nWe examined a total of 73 lesions from 27 individuals affected by endometriosis and focused on \nwithin patient heterogeneity – a largely overlooked feature amongst current genomic studies on \nendometriosis. Cases were specifically selected to have multiple anatomical sites affected as \nwell as multiple “types” of endometriosis:  EM, DIE and OMA. Using a high-sensitivity, and error-\ncorrecting, sequencing technology followed by validation and additional screening with ddPCR \nwe identified somatic cancer driver alterations in lesions from 13 patients. A total of 27 lesions \nhad at least one driver alteration, 7 lesions had more than 5. \nWe found evidence of identical alterations across lesions in 10 individuals, all of which spanned \nmore than one type of endometriosis. The relatively commonplace finding of identical mutations \nacross lesions suggests that at least a subset are clonal: sharing a common ancestor within a \ngiven patient. According to our present data set, the order/directionality of dissemination cannot \nbe determined.  \nIn 9 lesions there also appeared to be multiple functionally redundant alterations, which is \nconsistent with individual lesions being oligoclonal (i.e. comprised a small number of clones), \nand often spread across multiple anatomically distinct lesions. Functionally redundant mutations \nwere seen in PIK3CA (activating), CTNNB1 (stabilizing), KRAS (activating), and more generally \nactivating Ras-pathway (e.g. KRAS, NRAS, ERBB2, EGFR). Redundant mutation in the same \ngene were seen in 6 cases, half of which had the redundant alterations in two or more lesions. \nIntra-lesion Ras-pathway redundancy, including activating somatic alterations in the same gene \nor different Ras-pathway genes, affected 9 lesions in 5 cases.  \nExamining the specific prevalence of mutations across lesions we observed more PIK3CA \nmutations than any other. Amongst informative cases, OMAs tended to have more mutations, \nwith OMA lesions from cases 1, 2, 3 having multiple activating PIK3CA and/or CTNNB1 (and \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n12 \n \nothers) driving the average mutation burden up. In contrast, DIE appeared to have a wider \nspectrum of mutations with EGFR and ERBB2 mutations seen only in DIE. Despite these raw \nnumbers, more lesion and more individuals appeared to be affected by at least one Ras-\npathway activating alteration, most frequently KRAS.  \nOur study is unique in examination of a large number of co-existing lesions per patient, allowing \nus to analyze both molecular heterogeneity and potential clonal dissemination/metastasis. \nParticular strengths include enrichment of endometriosis glands and stroma, the use of proven \nhigh-sensitivity and specificity digital sequencing methods, and orthogonal validation for the \nmajority of alterations (13). \nDue to application of manual macrodissection over laser capture microdissection we did not \nisolate (stromal/epithelial) cell types and can only use the variant allele frequency for binary \npresence or absence assessment of somatic alterations. IHC observations suggested somatic \nalterations were exclusive to the epithelium, similar to prior reports (10, 11, 32). In addition, we \nused only a relatively limited panel assay to screen for mutations, albeit studies that have \nundertaken exome analysis of endometriosis have most frequently reported somatic alterations \nin genes where our mutation panel has coverage (10, 12, 32, 33). Nonetheless, while we can \nmake suggestions about potential lineage of samples with informative mutations, we are \nmissing considerable data about the un-analyzed genome. In these cases, panel sequencing \nand subsequent ddPCR often required re-sampling for validation. The fraction of enriched \nendometriosis cells harboring mutation may change as the specimen is sectioned/sampled, and \nthat it would genuinely yield a fraction of cells below a detection threshold in one sampling and \nabove a threshold in another. Thus, this should be considered as a minimum estimate of \nclonality. Lesions (within a given patient) that do not share mutations cannot be definitively \ndetermined to be of unrelated lineage.  \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n13 \n \nIn recent years there has been a shift away from seeing endometriosis as one disease entity. In \naddition to biochemical variations, considerable efforts are being applied to identify subtype-\nspecific genomic alterations. Our findings validate a modest spectrum of driver alterations, as \nhas been described previously (10, 12, 32, 33), while shared alterations between lesions \nsupport a model of metastasis. Our observations allowed us to partially categorize cases based \non mutation load and distribution: those without detectable mutations, simple, and complex \ncases. It is worthwhile noting that all complex cases, with higher mutation burden as well as \ngene and pathway redundancy, were stage IV while other categories showed a range including \nlow stage cases. Further, gene and pathway redundancy in observed mutations, suggest \nendometriosis lesions may be comprised of a small number of clones within the epithelium and \nthat metastasis is also oligoclonal: these clones may disperse and travel together. \nTo make significant impact the next generation of endometriosis-genomic studies must apply \nhigh stringency methods, with appropriate enrichment of tissues, orthogonal validation and/or \nerror correcting sequencing technologies, and most critically be coupled to large clinical data \nregistries conforming to accepted standards in phenotyping data (13) - such as those promoted \nby WERF-EPHect (34, 35). Early studies on somatic genomics of endometriosis have \nsuggested relatively few alterations per lesion (10, 12, 32, 36). However, this work suggests \nendometriosis may have moderate genomic complexity. Any future study wishing to correlate \ngenomic heterogeneity with the burden of disease (stage) and clinical phenotypic spectrum \nmust take this into account. Accurate assessment of somatic mutation profiles may require a \nsignificant fraction of lesions are excised and tested at surgery (rather than ablated without \nbiopsy).  \nThis work has also shown that OMAs had the highest potential for oligoclonality, while DIE \nlesions were associated with wider range of mutations. Considering previous work in the field \n(37-40), this is consistent with the concept that distinct microenvironments are important for \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n14 \n \nlesion forming and spreading. The ovarian microenvironment may provide permissive conditions \nfor expansion of multiple clones which could be associated with the higher risk of malignant \ntransformation (41). DIEs might be surrounded by a more restrictive microenvironment \nconditions resulting in fewer clones and lower malignant potential. As a potential example: \nredundant PIK3CA activating mutation were prevalent in OMAs. Such PIK3CA alterations are \ncommon in endometriosis-associated cancers, and appear necessary (in combination with \nARID1A loss of function) for the generation of a representative clear cell ovarian carcinomas \nmodel in mice (42). Thus, OMAs with multiple PIK3CA mutant clones may have elevated \nmalignant potential. In contrast, the extraovarian microenvironment restriction on malignant \npotential has been suggested as a potential mechanism for the generally favorable outcomes \nobserved in low-stage synchronous (yet metastatic) endometrioid ovarian and endometrial \ncarcinomas (43). \n \nConclusion  \nOur results provide conclusive data for two major features of endometriosis: first that the \ndisease has a high potential for molecular heterogeneity as shown by mutation profiles \nindicating oligoclonality, or at least clonal divergence. Second, endometriosis is capable of \nmetastasis and this is not restricted to current definitions of endometriosis types. These features \nconfirm that the current classification of endometriosis is limited. While our series is not \nsufficient to suggest which molecular features should be included in a novel classification, we \ncan unequivocally state future work should consider patient-wide endometriosis mutation \nprofiles – not single lesions. This may include ranking of allele frequency, clonality across \nlesions, mutation burden (or clonal burden), and anatomic types. Intra-lesion spatial \nheterogeneity (such as in a large OMA or deep nodules) and clonal burden also warrants study \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n15 \n \nespecially in relapse/persistent chronic disease and those associated with malignant \nprogression.  \n \nAcknowledgements \nThe authors thank all the study participants who contributed to this study. We further recognize \nthe invaluable contributions of Prof. Dr. Sara Y. Brucker for continuous support of international \ncollaborations between the University Hospital Tübingen and the University of British Columbia, \nas well as PD Dr. Annette Staebler and the staff of the Institute of Pathology, University Hospital \nTübingen for facilitating access to pathology archives.  \nFunding was provided by Canadian Institutes of Health Research (Early Career Investigator \nGrant in in Maternal, Reproductive, Child & Youth Health to MS Anglesio). M Köbel received \nsupport through the Calgary Laboratory Services research support fund (RS19-609). MS \nAnglesio is funded through a Michael Smith Foundation for Health Research Scholar Award \n(18274) and the Janet D. Cottrelle Foundation Scholars program (managed by the BC Cancer \nFoundation). This project received technical and data management support from Calgary \nLaboratory Services and the Genetic Pathology Evaluation Centre (GPEC). GPEC receive core \nsupport from BC's Gynecological Cancer Research team (OVCARE), and the VGH+UBC \nHospital Foundation. M Köbel was supported by internal research support (RS19-621).  \nConflict of Interest Declaration \nThe authors declare no competing or conflicting interests, financial or personal. No funder had \nany role in the study design, collection of data, recruitment or participants, interpretation of \nresults, manuscript content, or decision to publish.   \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n16 \n \nReferences \n \n1. Munksgaard PS, Blaakaer J. 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Prognostic and \nTheranostic Biomarkers in Ovarian Clear Cell Carcinoma. Int J Gynecol Pathol 2021. \n31. Kang EY, Cheasley D, LePage C, Wakefield MJ, da Cunha Torres M, Rowley S  et al. \nRefined cut-off for TP53 immunohistochemistry improves prediction of TP53 mutation status in \novarian mucinous tumors: implications for outcome analyses. Mod Pathol 2021;34:194-206. \n32. Noe M, Ayhan A, Wang TL, Shih IM. Independent development of endometrial \nepithelium and stroma within the same endometriosis. J Pathol 2018. \n33. Inoue S, Hirota Y, Ueno T, Fukui Y, Yoshida E, Hayashi T  et al. Uterine adenomyosis is \nan oligoclonal disorder associated with KRAS mutations. Nat Commun 2019;10:5785. \n34. Fassbender A, Rahmioglu N, Vitonis AF, Vigano P, Giudice LC, D'Hooghe TM  et al. \nWorld Endometriosis Research Foundation Endometriosis Phenome and Biobanking \nHarmonisation Project: IV. Tissue collection, processing, and storage in endometriosis research. \nFertility and sterility 2014;102:1244-53. \n35. Rogers PA, Adamson GD, Al-Jefout M, Becker CM, D'Hooghe TM, Dunselman GA  et al. \nResearch Priorities for Endometriosis. Reprod Sci 2017;24:202-26. \n36. Bulun SE, Wan Y, Matei D. Epithelial Mutations in Endometriosis: Link to Ovarian \nCancer. Endocrinology 2019;160:626-38. \n37. Artemova D, Vishnyakova P, Khashchenko E, Elchaninov A, Sukhikh G, Fatkhudinov T. \nEndometriosis and Cancer: Exploring the Role of Macrophages. Int J Mol Sci 2021;22. \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n18 \n \n38. Olkowska-Truchanowicz J, Bialoszewska A, Zwierzchowska A, Sztokfisz-Ignasiak A, \nJaniuk I, Dabrowski F et al. Peritoneal Fluid from Patients with Ovarian Endometriosis Displays \nImmunosuppressive Potential and Stimulates Th2 Response. Int J Mol Sci 2021;22. \n39. Oda K, Hamanishi J, Matsuo K, Hasegawa K. Genomics to immunotherapy of ovarian \nclear cell carcinoma: Unique opportunities for management. Gynecologic oncology \n2018;151:381-9. \n40. Lai CR, Hsu CY, Chen YJ, Yen MS, Chao KC, Li AF. Ovarian cancers arising from \nendometriosis: a microenvironmental biomarker study including ER, HNF1ss, p53, PTEN, \nBAF250a, and COX-2. Journal of the Chinese Medical Association : JCMA 2013;76:629-34. \n41. Saavalainen L, Lassus H, But A, Tiitinen A, Harkki P, Gissler M  et al. Risk of \nGynecologic Cancer According to the Type of Endometriosis. Obstet Gynecol 2018. \n42. Chandler RL, Damrauer JS, Raab JR, Schisler JC, Wilkerson MD, Didion JP  et al. \nCoexistent ARID1A-PIK3CA mutations promote ovarian clear-cell tumorigenesis through pro-\ntumorigenic inflammatory cytokine signalling. Nat Commun 2015;6:6118. \n43. Anglesio MS, Wang YK, Maassen M, Horlings HM, Bashashati A, Senz J  et al. \nSynchronous Endometrial and Ovarian Carcinomas: Evidence of Clonality. J Natl Cancer Inst \n2016;108. \n  \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n19 \n \nFigure Legends \nFigure 1: Fraction of cohort affected by somatic hotspot mutations detected in panel \nsequencing. (A) Overall split of cases with at least one lesions having at least one somatic \ncancer-driver alteration. (B) Numbers of detected somatic mutations, split by affected gene, \nacross our entire cohort. (C) Fraction of mutation affected lesions, split by mutation type. (D) \nFraction of mutation affected lesions, split by lesion type. (E) Fraction of mutation affected \nlesions, summarized by fraction of affected cases. \nFigure 2: Clonal relationships in Case 3 patient presenting with DIE of the vagina, DIE of the \ncul-de-sac, an OMA of the left ovary, and EM of the rectum. (A) Detected somatic alteration \nfrom sequencing and mutation surrogate IHC suggest identical/clonal alteration in KRAS, \nERBB2, and PIK3CA (p.Met1043Ile) between OMA and two anatomically distinct lesions of DIE. \nAdditional PIK3CA alterations are also visible in the OMA and vaginal DIE. Heterogeneous loss \nof ARID1A is observed in only the OMA and EM (insufficient material was available for mutation \ntesting in EM). (B) Three hypothetical dissemination models explaining the mutational pattern: \n(1) top panel illustrates a linear pattern wherein clones from the cul-de-sac travel to the vagina \nand acquired additional alterations in PIK3CA. Subsequent transfer of all clones to the ovary \nwhere subclonal-ARID1A loss occurs. It may further be speculated that the EM resulted from a \ntransfer of clones between the vaginal DIE and OMA (as the initial site for ARID1A loss) or \nsubsequent spread after clones established on the ovary. Given the lack of mutational data on \nthe EM lesion it may also be entirely independent. (2) middle panel illustrates an example where \na complex set of clones exist at the ovary and only a subset of these break-off and colonize \nvaginal, cul-de-sac and EM/rectal sites. Finally, (3) Lower panel illustrates parallel dissemination \nfrom the cul-de-sac lesion to both the vaginal and ovarian sites. Herein we may consider sub-\nthreshold signal from ddPCR of p.Arg88Gln, p.Glu545Lys and p.Gly1049Ser PIK3CA alteration \n(Table S2) as weak evidence of emerging/undetectable clones that subsequently expand post-\n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n20 \n \ntransfer of cells to both vagina and ovary. In this model, the OMA acquires loss-of function \nARID1A alterations. Additional possibilities may also explain the mutational patterns. (C-D-E) \nphotomicrographs showing ARID1A IHC results including regions of loss (surrogate for loss-of \nfunction mutation; black arrows) and normal (retained nuclear staining; red arrows) in \nendometriosis epithelium. \nFigure 3: Clonal relationships in Case 4 patient presenting with DIE of the rectum, EM of the \ncul-de-sac and an OMA on the left ovary. (A) Detected somatic alteration from sequencing \nsuggest all variants are shared between DIE and EM lesions. In contrast the ovarian lesion \nshares only 1 PIK3CA and 1 KRAS alteration with the other two and has no (detected) unique \nchanges. (B) Three possible dissemination models explaining the mutational pattern: (1) top \npanel illustrates a linear model wherein clones from the ovary travel to the rectum, expand and \nacquire additional alteration. Cells from the DIE containing all clones then seed the EM on the \nCul-de-sac. (2) middle panel illustrates another linear model of dissemination wherein a \ncomplex clonal population at the Rectum seed the cul-de-sac. Subsequently only a subset of \nthese clone travel to and populate the ovary. Finally, (3) illustrates a differing model of spread \nwherein a complex clonal population at the Rectum seed the cul-de-sac with all clones. Only a \nsubset with a single PIK3CA and KRAS altered clone break-off, or are capable of, colonizing the \novary. \n \n  \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n21 \n \nFigures \nA B \nC D \nE \n0\n0,05\n0,1\n0,15\n0,2\n0,25\n0,3\n0,35\n0,4\nPIK3CA NRAS CTNNB1 KRAS ERBB2 EGFR\nFraction of affected lesions, split by lesion type\nOMA DIE EM\nFigure 1: Fraction of cohort affected by somatic hotspot mutations detected in panel sequencing. \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n22 \n \n  A B\nC D E \nCase 3 (OMA) Case 3 (OMA) \nCase 3 (EM) \nA p.G ly 1049SerX X\nK3CA p.Arg88Gln XX\nK3CA p.Met1043Ile XX X\nRAS p.G ly 12Asp X X X\nRBB2 p.Ser310Phe XX X\nRID 1A\nC \nLos s  in aty pic al\nendom etrios is \nHeterogeneous \nlos s \nPI\nPI\nK\nE\nA\nIH\nCase 3 Detected Alterations\nGene Hotspot OMA\nLeft ovary\nDIE (v)\nVagina\nDIE (c)\nCul-de-sac\nEM\nRectum\nPIK3CA p.Glu545Lys X X Insufficient\nsize for\nsequencing\nPIK3CA p.Gly1049Ser X X\nPIK3CA p.Arg88Gln X X\nPIK3CA p.Met1043Ile X X X\nKRAS p.Gly12Asp X X X\nERBB2 p.Ser310Phe X X X\nARID1A\nIHC\nLoss in \natypical\nendometriosis\nHetero-\ngeneous\nloss\nD IE(c)\nC ul-de-s ac \nDI E (v) Vagina\nOM A\nLef t ovary \nDI E(c)\nCul-de-Sac \nDIE  (v)  Vagina\nOMA \nLefto v ary\n(1) \n(3) \n(2) \nOMA \nLeft ovary \nOMA \nRectum DIE(c) \nCul-de-sac \nDIE (v) \nVagina \n+? \nFigure 2: Clonal relationships in Case 3 patient presenting with DIE of the vagina, DIE of the cul-de-sac, \nan OMA of the left ovary, and EM of the rectum. \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n23 \n \n  \nCase 4 DetectedAlterations\nGene Hotspot DIE\nRectum\nOMA\nLeft ovary\nEM\nCul-de-sac\nPIK3CA p.Glu542Lys X X\nPIK3CA p.Glu545Lys X X\nPIK3CA p.Gly1049Ser X X\nPIK3CA p.Met1043Ile X X X\nNRAS p.Gly13Asp X X\nCTNNB1 p.His36T yr X X\nCTNNB1 p.Gly34Glu X X\nKRAS p.Gly12Asp X X X\n(3) \nA B (1)\n(2) \nOMA\nLeft ovary\nDIE \nRectum\nEM\nCul-de-sac\nDIE \nRectum\nEM\nCul-de-sac\nOMA\nLeft ovary\nEM\nCul-de-sac\nOMA\nLeft ovary\nDIE \nRectum\nX \nX \nFigure 3: Clonal relationships in Case 4 patient presenting with DIE of the rectum, EM of the cul-de-sac \nand an OMA on the left ovary. \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n24 \n \n \nTables \nTable 1: Clinical characteristics of endometriosis cases categorized by mutation profile.  \n  Full Cohort Wild-type \ncase subset \nSimple case \nsubset \nComplex \ncase subset \nPatient Profile \nn 27 14 7 6 \nAge (range) 34.85 (23-45)  34.5 (23-45)  34.29 (25-40)  36.33 (31-44)  \nStage I  2 1 1 0 \nStage I I  3 2 1 0 \nStage I II  6 4 2 0 \nStage IV  16 7 3 6 \nNum\nbers of Lesion\ns \nOMA lesio ns 17 (1.06)  8 (1)  3 (1.5)  6 (1)  \nOMA in pan el  14 (1.08)  6 (1)  3 (1.5)  5 (1)  \nDIE lesions 38 (1.41)  18 (1.29)  13 (1.86)  7 (1.17)  \nDIE in panel 29 (1.26)  12 (1)  12 (1.71)  5 (1.25)  \nEM lesions 18 (1.13)  8 (1.14)  5 (1)  5 (1.25)  \nEM in panel  10 (1)  5 (1)  4 (1)  1 (1)  \nSurgical Ind\nication \nPain (only)  13 9 2 2 \nFertili ty (only)  9 3 3 3 \nPain and Fertili ty 3 2 1 0 \nunknown 1 0 0 1 \nother *  1 0 1 0 \nPatient Profile summarizes data on the age and diagnosis of the patients, including the average age of \nper category and range (in brackets). Note wild-type cases subset are those that did not have detectable \nsomatic alteration in panel sequencing. Numbers of Lesions summarizes the total number of lesions per \ncategory as well as the number included in our panel sequencing (in panel). The number in brackets \nrefers to an average number of lesions per affected patient in the given category. Surgical Indications is \nbased the reported symptoms from surgical report (*other: hematochezia, incidental finding of \nendometriosis with no report of pain or fertility issue). Abbreviations: DIE= Deep infiltrating endometriosis, \nEM= superficial endometriosis, OMA= endometrioma. For additional detail see Table S4. \n  \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n25 \n \nSupplemental Figure and Table Legends  \nFigure S1: Project workflow. 27 patients were selected all with more than one type and more \nthan one anatomical site of endometriosis. A total of 73 lesions were reviewed for analysis. 53 \nlesions had sufficient tissue for digital panel sequencing while an additional 6 samples had \nsufficient yield for only ddPCR of select alterations.  A total of 83 somatic alterations were \ninitially detected in sequencing, 72 subjected to ddPCR validation (58 validated). During the \nddPCR validation stage additional lesion were tested for alterations observed in panel \nsequencing resulting in a total of 90 detected alterations in 13 cases across the entire cohort.  \nImmunohistochemistry for PTEN (loss in 11%), ARID1A (loss in 3%), and p53 (no abnormal \nstaining observed) was done in parallel.  \nFigure S2: Per-Case summary of detected alterations across lesions as derived from \nsequencing, digital droplet PCR (ddPCR), and immunohistochemistry (IHC) in informative \ncases. Legend: Abd = abdominal, atyp= atypical, DIE= deep infiltrating endometriosis, EM= \nsuperficial endometriosis, Het loss= heterogeneous loss, l= left, lig= ligament, \npararec=pararectal, OMA= endometrioma, r=right rv= rectovaginal, su= sacrouterine   \nTable S1: Hotspots and Exons analyzed in the FIND IT\nTM panel version 3.4 (Canexia Health, \nCanada)  \nTable S2A: Panel Sequencing and ddPCR validation data. Full list of sequence and droplet \ndigital PCR (ddPCR) data with variant allele frequencies (VAFs) and probability scores (PR.)   \nDIE= deep infiltrating endometriosis, EM= superficial endometriosis, OMA= endometrioma  \nTable S2B: Immunohistochemistry. Full list of IHC results, DIE= deep infiltrating endometriosis, \nEM= superficial endometriosis, OMA= endometrioma  \nTable S3: Resource table of antibodies, primers, and probes \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint \n\n  \n \n26 \n \nTable S4: Cohort Overview. Each cell shows the number of lesions available and tested from \nthe noted anatomical site/type (number in brackets denote lesions were not tested in panel \nsequencing and detected only in ddPCR). Blue cell = shared alteration within case/between \nlesions marked by blue cells in table (cells not in blue were uninformative/did not show evidence \nof clonality with any other lesion from the given case), Grey lines/cases were entirely \nuninformative (no mutation detected in panel sequencing from any tested lesion). Abbreviations: \nDIE= Deep infiltrating endometriosis, EM= superficial endometriosis, OMA= endometrioma \n . CC-BY-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 30, 2021. ; https://doi.org/10.1101/2021.04.12.21255355doi: medRxiv preprint","source_license":"CC0","license_restricted":false}