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Clinical and molecular characterization of ovarian carcinoma displaying isolated lymph node relapse

Open AccessPublished:May 02, 2019DOI:https://doi.org/10.1016/j.ajog.2019.04.035

      Background

      Disease relapse is the primary cause of death from ovarian carcinoma. Isolated lymph node relapse is a rare pattern of ovarian carcinoma recurrence, with a reported median postrelapse survival of 2.5 to 4 years. To date, investigations have not compared isolated lymph node relapse ovarian carcinoma directly to a matched extranodal relapse cohort or performed molecular characterization of cases that subsequently experience isolated lymph node relapse.

      Objective

      Here we seek to compare the clinical outcome, tumor-infiltrating lymphocyte burden, and frequency of known prognostic genomic events in isolated lymph node relapse ovarian carcinoma vs extranodal relapse ovarian carcinoma.

      Study Design

      Forty-nine isolated lymph node relapse ovarian carcinoma patients were identified and matched to 49 extranodal relapse cases using the Edinburgh Ovarian Cancer Database, from which the clinical data for identified patients were retrieved. Matching criteria were disease stage, histologic subtype and grade, extent of residual disease following surgical debulking, and age at diagnosis. Clinicopathologic factors and survival data were compared between the isolated lymph node relapse and extranodal relapse cohorts. Genomic characterization of tumor material from diagnosis was performed using panel-based high-throughput sequencing and tumor-infiltrating T cell burden was assessed using immunohistochemistry for CD3+ and CD8+ cells.

      Results

      Isolated lymph node relapse cases demonstrated significantly prolonged postrelapse survival and overall survival vs extranodal relapse upon multivariable analysis (HRmulti = 0.52 [0.33–0.84] and 0.51 [0.31–0.84]). Diagnostic specimens from high-grade serous ovarian carcinomas that subsequently displayed isolated lymph node relapse harbored significantly greater CD3+ and CD8+ cell infiltration compared to extranodal relapse cases (P = .001 and P = .009, Bonferroni-adjusted P = .003 and P = .019). Isolated lymph node relapse high-grade serous ovarian carcinoma cases did not show marked enrichment or depletion of cases with BRCA1/2 mutation or CCNE1 copy number gain when compared to their extranodal relapse counterparts (24.4% vs 19.4% and 18.2% vs 22.6%, P = .865 and P = .900).

      Conclusion

      Isolated lymph node relapse ovarian carcinoma represents a distinct clinical entity with favorable outcome compared to extranodal relapse. There was no clear enrichment or depletion of BRCA1/2 mutation or CCNE1 gain in the isolated lymph node relapse ovarian carcinoma cohort compared with extranodal relapse cases, suggesting that these known prognostic genomically defined subtypes of disease do not display markedly altered propensity for isolated lymph node relapse. Diagnostic tumor material from isolated lymph node relapse patients demonstrated greater CD3+ and CD8+ cell infiltration, indicating stronger tumor engagement by T cell populations, which may contribute to the more indolent disease course of isolated lymph node relapse.

      Key words

      Ovarian carcinoma (OC) is the most lethal gynecologic malignancy, accounting for over 180,000 deaths per year worldwide.
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      OC is now recognized to comprise 5 core histologic subtypes: high-grade serous (HGS), endometrioid, clear cell, low-grade serous, and mucinous OC—each displaying distinct molecular landscapes and clinical behavior.
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      Conversely, CCNE1 copy number gain has been associated with chemoresistance and poorer survival in this group.
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      Integrated genomic analyses of ovarian carcinoma.
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      Why was this study conducted?

      • A number of investigators have reported a relatively indolent disease course in ovarian carcinoma patients experiencing isolated lymph node relapse. However, none have systematically compared these to extranodal relapse or performed molecular characterization of patients who go on to experience this distinct pattern of recurrence.

      Key findings

      • Isolated lymph node relapse patients demonstrated significantly prolonged overall and postrelapse survival compared with extranodal relapse cases. Isolated lymph node relapse cases demonstrated greater tumor-infiltrating lymphocyte burden at diagnosis, but did not demonstrate significant enrichment or depletion of BRCA1/2 mutation or gain of CCNE1, both known to be prognostic in ovarian carcinoma.

      What does this add to what is known?

      • This is the first report demonstrating significantly improved clinical outcome in isolated lymph node relapse ovarian carcinoma when compared directly with extranodal relapse, and represents the first study to perform molecular characterization of patients who go on to experience isolated lymph node relapse.
      Though patients in most OC cases—particularly HGS OCs—are typically sensitive to chemotherapy in the first-line setting, the majority of patients will experience disease relapse, which acquires resistance to chemotherapy.
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      Involvement of lymph nodes (LNs) at relapse is common; however, recurrence confined solely to LNs is a rare event, accounting for ≤5% of relapsed OCs.
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      Epithelial ovarian cancer relapsing as isolated lymph node disease: natural history and clinical outcome.
      These isolated LN relapse (ILNR) cases have been described as a unique clinical disease entity and are thought to experience a relatively indolent disease course, with a reported median postrelapse survival (PRS) and overall survival (OS) of around 2.5 to 4 years and >5 years, respectively.
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      Isolated lymph node relapse of epithelial ovarian carcinoma: outcomes and prognostic factors.
      • Legge F.
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      Epithelial ovarian cancer relapsing as isolated lymph node disease: natural history and clinical outcome.
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      Outcomes after combined therapy including surgical resection in patients with epithelial ovarian cancer recurrence(s) exclusively in lymph nodes.
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      • Li Z.
      • Feng Y.L.
      • Liu J.H.
      [Treatment and prognostic analysis of ovarian cancer patients with isolated region of lymph node recurrence].
      • Ferrero A.
      • Ditto A.
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      • et al.
      Secondary cytoreductive surgery for isolated lymph node recurrence of epithelial ovarian cancer: a multicenter study.
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      • Karam A.K.
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      • et al.
      Secondary cytoreductive surgery for isolated nodal recurrence in patients with epithelial ovarian cancer.
      • Fotiou S.
      • Aliki T.
      • Petros Z.
      • et al.
      Secondary cytoreductive surgery in patients presenting with isolated nodal recurrence of epithelial ovarian cancer.
      A number of previous studies have reported on the clinical outcome of apparent ILNR OC (summarized in Table 1).
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      • Plantade A.
      • Pages C.
      • et al.
      Isolated lymph node relapse of epithelial ovarian carcinoma: outcomes and prognostic factors.
      • Legge F.
      • Petrillo M.
      • Adamo V.
      • Pisconti S.
      • Scambia G.
      • Ferrandina G.
      Epithelial ovarian cancer relapsing as isolated lymph node disease: natural history and clinical outcome.
      • Uzan C.
      • Morice P.
      • Rey A.
      • et al.
      Outcomes after combined therapy including surgical resection in patients with epithelial ovarian cancer recurrence(s) exclusively in lymph nodes.
      • Gadducci A.
      • Cosio S.
      • Zola P.
      • et al.
      The clinical outcome of epithelial ovarian cancer patients with apparently isolated lymph node recurrence: a multicenter retrospective Italian study.
      • Tu H.
      • Huang H.
      • Huang Q.D.
      • Li Z.
      • Feng Y.L.
      • Liu J.H.
      [Treatment and prognostic analysis of ovarian cancer patients with isolated region of lymph node recurrence].
      • Ferrero A.
      • Ditto A.
      • Giorda G.
      • et al.
      Secondary cytoreductive surgery for isolated lymph node recurrence of epithelial ovarian cancer: a multicenter study.
      • Santillan A.
      • Karam A.K.
      • Li A.J.
      • et al.
      Secondary cytoreductive surgery for isolated nodal recurrence in patients with epithelial ovarian cancer.
      • Fotiou S.
      • Aliki T.
      • Petros Z.
      • et al.
      Secondary cytoreductive surgery in patients presenting with isolated nodal recurrence of epithelial ovarian cancer.
      Many of these studies have reported only a small number of cases,
      • Blanchard P.
      • Plantade A.
      • Pages C.
      • et al.
      Isolated lymph node relapse of epithelial ovarian carcinoma: outcomes and prognostic factors.
      • Uzan C.
      • Morice P.
      • Rey A.
      • et al.
      Outcomes after combined therapy including surgical resection in patients with epithelial ovarian cancer recurrence(s) exclusively in lymph nodes.
      • Santillan A.
      • Karam A.K.
      • Li A.J.
      • et al.
      Secondary cytoreductive surgery for isolated nodal recurrence in patients with epithelial ovarian cancer.
      • Fotiou S.
      • Aliki T.
      • Petros Z.
      • et al.
      Secondary cytoreductive surgery in patients presenting with isolated nodal recurrence of epithelial ovarian cancer.
      with a minority reporting larger numbers identified from multiple centers.
      • Gadducci A.
      • Cosio S.
      • Zola P.
      • et al.
      The clinical outcome of epithelial ovarian cancer patients with apparently isolated lymph node recurrence: a multicenter retrospective Italian study.
      • Ferrero A.
      • Ditto A.
      • Giorda G.
      • et al.
      Secondary cytoreductive surgery for isolated lymph node recurrence of epithelial ovarian cancer: a multicenter study.
      To our knowledge, none of these studies have compared outcome directly to a matched extranodal relapse (ENR) cohort. Furthermore, they have not performed contemporary histologic subtyping or molecular characterization so as to identify potential subgroups of disease with a propensity to experience this distinct pattern of disease relapse.
      Table 1Previous reports of isolated lymph node relapse ovarian carcinoma
      ILNR casesNFerrero
      • Ferrero A.
      • Ditto A.
      • Giorda G.
      • et al.
      Secondary cytoreductive surgery for isolated lymph node recurrence of epithelial ovarian cancer: a multicenter study.
      Tu
      • Tu H.
      • Huang H.
      • Huang Q.D.
      • Li Z.
      • Feng Y.L.
      • Liu J.H.
      [Treatment and prognostic analysis of ovarian cancer patients with isolated region of lymph node recurrence].
      Gadducci
      • Gadducci A.
      • Cosio S.
      • Zola P.
      • et al.
      The clinical outcome of epithelial ovarian cancer patients with apparently isolated lymph node recurrence: a multicenter retrospective Italian study.
      Fotiou
      • Fotiou S.
      • Aliki T.
      • Petros Z.
      • et al.
      Secondary cytoreductive surgery in patients presenting with isolated nodal recurrence of epithelial ovarian cancer.
      Legge
      • Legge F.
      • Petrillo M.
      • Adamo V.
      • Pisconti S.
      • Scambia G.
      • Ferrandina G.
      Epithelial ovarian cancer relapsing as isolated lymph node disease: natural history and clinical outcome.
      Santillan
      • Santillan A.
      • Karam A.K.
      • Li A.J.
      • et al.
      Secondary cytoreductive surgery for isolated nodal recurrence in patients with epithelial ovarian cancer.
      Blanchard
      • Blanchard P.
      • Plantade A.
      • Pages C.
      • et al.
      Isolated lymph node relapse of epithelial ovarian carcinoma: outcomes and prognostic factors.
      Uzan
      • Uzan C.
      • Morice P.
      • Rey A.
      • et al.
      Outcomes after combined therapy including surgical resection in patients with epithelial ovarian cancer recurrence(s) exclusively in lymph nodes.
      Summary
      7338692132252712Few reports of ≥40 cases
      Age at diagnosisYearsMedian 5424 (63%) >50; 14 (37%) ≤50Median 58Mean 50Median 60Mean 58Mean 59Median 51Largely unremarkable compared with unselected OC cohorts
      Range29–7334–7836–6745–7641–8241-8542–71
      Stage at diagnosisI14 (19%)011 (16%)3 (14%)02 (8%)4 (15%)5 (42%)
      II4 (6%)15 (39%)6 (9%)3 (14%)1 (3%)5 (20%)5 (18%)1 (8%)
      III51 (70%)23 (61%)46 (67%)14 (67%)29 (91%)15 (60%)15 (56%)6 (50%)
      IV4 (6%)06 (9%)1 (5%)2 (6%)3 (12%)3 (11%)0
      RD following first-line debulking0 cm / <0.5 cm57 (78%)17 (45%)22 (32%)8 (38%)14 (44%)18 (72%)NA7 (58%)
      ≤1 cm10 (14%)10 (26%)11 (16%)7 (33%)6 (19%)5 (20%)NA4 (33%)
      <2 cm4 (6%)11 (29%)36 (52%)4 (19%)0NA
      >2 cm2 (3%)2 (10%)12 (38%)2 (8%)NA1 (8%)
      Grade at diagnosisI4 (6%)7 (18%)3 (4%)09 (32%)25 (100%) high gradeNANA
      II5 (7%)14 (37%)13 (19%)8 (38%)
      III64 (88%)17 (45%)54 (78%)13 (62%)19 (68%)
      NA----4-
      Reported histologic subtype at diagnosisSerous53 (73%)19 (50%)52 (75%)16 (76%)26 (81%)19 (76%)17 (62%)
      Includes 5 cases described as papillary
      8 (67%)Predominantly serous / HGS cases, as with unselected OC cohorts
      Endometrioid11 (15%)9 (24%)12 (17%)5 (24%)2 (6%)2 (8%)3 (11%)3 (25%)
      Clear cell001 (1%)0000
      Mucinous1 (1%)1 (3%)01 (3%)03 (11%)0
      Other8 (11%)9 (24%)4 (6%)3 (9%)4 (16%)4 (15%)1 (8%)
      DFI / time to ILNR
      From end of first-line chemotherapy.
      Median months181844 (62%)

      >12 months
      2117.51626 months from diagnosis21Median 1.5–2 years DFI
      Range6–1929-968–1561–1346–401–1596–72
      ILNR site(s)Para-aortic only37 (51%)10 (26%)23 (33%)8 (38%)14 (44%)15 (60%)9 (33%) retro. alone, 6(22%) retro. + other. supraclavicular, mediastinal, iliac, and inguinal involvement in 7 (26%), 4 (15%), 4 (15%), and 3 (11%) cases5 (42%)Most commonly involves pelvic and/or para-aortic sites
      Pelvic only21 (29%)15 (39%)12 (17%)4 (19%)1 (3%)3 (12%)4 (33%)
      Para-aortic & pelvic9 (12%)7 (18%)6 (9%)4 (19%)9 (28%)1 (4%)1 (8%)
      Inguinal only3 (4%)2 (5%)12 (17%)4 (19%)2 (6%)5 (20%)1 (8%)
      Other combinations3 (4%)4 (11%)16 (23%)1 (5%)6 (19%)1 (4%)1 (8%)
      ILNR patternSingle region61 (84%)27 (71%)47 (77%)17 (81%)20 (63%)24 (96%)17 (63%)10 (83%)Most commonly localized to a single region
      Multiregion12 (16%)11 (29%)14 (23%)4 (19%)9 (28%)1 (4%)10 (37%)2 (17%)
      NA--8-3---
      PRSMedian months5-y PRS 64%; 5-y OS ∼80%5-y PRS 66.5%32.1473737265-y PRS 71%Median 2–4 years
      OSMedian months62.9661096168Median >5 years
      Surgery for ILNRYes73 (100%)19 (50%)24 (35%)21 (100%)12 (38%)25 (100%)8 (30%)12 (100%)Heterogeneous management, typically involving chemotherapy
      No019 (50%)45 (65%)020 (63%)019 (70%)0
      ILNR intervention: regimeChemo alone05 (13%)44 (64%)019 (59%)08 (30%)0
      Surgery alone3 (4%)01 (1%)01 (3%)2 (8%)2 (7%)0
      Surgery-chemo combination70 (96%)19 (50%)22 (32%)17 (81%)11 (34%)15 (60%)5 (19%)10 (83%)
      Radio alone001 (1%)0002 (7%)0
      No intervention00001 (3%)07 (26%)0
      Other014 (37%)1 (1%)4 (19%)08 (32%)3 (11%)2 (17%)
      Chemo, chemotherapy; DFI, disease-free interval; ENR, extranodal relapse; ILNR, isolated lymph node relapse; NA, not available; OS, overall survival; PRS, postrelapse survival; radio, radiotherapy; RD, residual disease; retro, retroperitoneal.
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      a Includes 5 cases described as papillary
      b From end of first-line chemotherapy.
      Here, we report clinical and molecular characterization of a matched ILNR and ENR cohort with contemporary pathology review to compare the clinical outcome and molecular landscape of ILNR and ENR OC.

      Materials and Methods

      Isolated lymph node relapse patient identification

      ILNR OC cases were identified from the Edinburgh Ovarian Cancer Database (Appendix: Supplementary Figure S1), wherein the clinical variables, treatment details, and follow-up data of OC patients treated within the Edinburgh Cancer Centre are collected prospectively as part of routine care. Potential ILNR cases were identified using the search terms “lymph node” or “groin node” as the dominant site of relapse, yielding 161 results. Nonepithelial tumors (n = 1), tumors of borderline malignancy (n = 1), and primary LN serous carcinomas (n = 2) were excluded. Patients with concurrent extranodal disease (n = 50), lack of cross-sectional imaging to confirm sole ILNR (n = 13), or coexistence of other malignancies leading to uncertain origin of LN disease (n = 2) were excluded. Patients with residual disease (RD) after completion of first-line treatment (n = 19) or insufficient clinical data for eligibility assessment (n = 24) were also excluded, leaving 49 ILNR cases.

      Matching of isolated lymph node relapse to extranodal relapse

      ILNR cases were electronically matched to ENR cases with complete response to first-line therapy using the Edinburgh Ovarian Cancer Database. Matching criteria were as follows: (1) diagnostic histologic subtype and grade, (2) stage at diagnosis, (3) extent of RD following debulking surgery, and (4) closest age at diagnosis following matching of criteria 1–3. Criteria were relaxed to facilitate matching of all ILNR cases as detailed in Supplementary Table S1 (Appendix).

      Ethical approval and tissue collection

      Clinical research access and ethical approval for correlation of molecular data to clinicopathologic features and clinical outcome in OC was obtained via NHS Lothian Research and Development (reference ID 2007/W/ON/29). Ethical approval for the use of human tumor material in translational research was obtained from South East Scotland Human Annotated Bioresource (Lothian NRS Bioresource Ethics Reference 15/ES/0094-SR831). Tumor material was available for 75.5% (74 of 98) of cases (77.6%, 38 of 49 ILNR and 73.5%, 36 of 49 ENR).

      Histologic subtyping of ovarian carcinomas

      Contemporary pathology review of ILNR and matched ENR cases was performed by an expert gynecologic pathologist (C.S.H.). Where appropriate (n = 9), immunohistochemistry (IHC) for WT1 and p53 was performed to aid histologic subtyping.
      • Kobel M.
      • Rahimi K.
      • Rambau P.F.
      • et al.
      An immunohistochemical algorithm for ovarian carcinoma typing.
      WT1 IHC was performed using 1:1000 dilutions of antibody M3561 clone 6F-H2 (Dako, Agilent Technologies, Santa Clara, CA). p53 staining was performed using 1:50 dilutions of antibody M7001 clone DO-7 (Dako, Agilent Technologies). Both stains were performed using the Leica Bond III Autostainer (Leica Biosystems, Milton Keynes, UK).

      Nucleic acid isolation

      Up to 10 10-μm formalin-fixed paraffin-embedded sections, macrodissected using marked hematoxylin–eosin-stained slides as a guide to enrich for tumor purity (Appendix: Supplementary Table S2), were used for DNA extraction. DNA was extracted using the QIAamp DNA FFPE Tissue Kit and Deparaffinization Solution (Qiagen, Venlo, the Netherlands).

      Panel-based sequencing of BRCA and non-BRCA homologous recombination deficiency genes

      High-throughput sequencing was performed using an 83-gene custom Integrated DNA Technologies gene capture panel with unique molecular indices, as described in the Appendix. Gene targets, centered around the homologous recombination DNA repair pathway, are detailed in Supplementary Table S3 (Appendix). The median per-sample mean target coverage achieved was 386X.

      Assessment of CCNE1 copy number

      Copy number variants in CCNE1 were characterized by TaqMan Genotyping qPCR Copy Number Assays (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA), as detailed in the Appendix.

      Assessment of tumor-infiltrating lymphocyte density

      Tumor-infiltrating lymphocytes (TILs) were assessed using 4-μm FFPE sections of diagnostic tumor material from first-line cytoreductive surgery. IHC for CD3 and CD8 was performed using Bond ready-to-use CD8-4B11 and CD3-LN10 antibodies (Leica Biosystems) on the Leica Bond III Autostainer. Human tonsil was used as a positive control for both markers. Stained slides were digitized and marker-positive cells were quantified using QuPath
      • Bankhead P.
      • Loughrey M.B.
      • Fernández J.A.
      • et al.
      QuPath: Open source software for digital pathology image analysis.
      in 8 randomly selected tumor-containing 500 × 500-μm fields per sample. Tumor area was marked as a region of interest (Appendix: Supplementary Figure S2) and marker-positive cells were quantified using the positive cell detection protocol as a percentage of the total cell number demonstrating marker positivity.
      TIL scoring validation was performed by manual counting of marker-positive cells by 2 human observers (R.L.H. and A.H.P.), in a randomly selected validation cohort representing 15% of samples for each marker. The correlation of marker-positive cell counts (observer 1 vs observer 2 vs QuPath) demonstrated excellent agreement for both markers (Spearman’s rho > 0.95, P < .0001 for all comparisons).

      Statistical analyses

      Statistical analyses were performed using R version 3.5.1 (R Foundation, Vienna, Austria). Disease-free interval (DFI) was calculated as time from end of first-line chemotherapy to disease recurrence. Comparisons of OS and PRS were conducted using Cox proportional hazards regression models within the Survival R package

      Therneau T. A Package for Survival Analysis in S. 2015. Available at: https://CRAN.R-project.org/package=survival. Accessed June 14, 2019.

      and presented as hazard ratios (HRs) alongside their 95% confidence intervals (CIs). Frequency comparisons were made using the χ2 test and Fisher exact test as appropriate. Comparisons of TIL density were made using the Mann–Whitney U test. Analyses were adjusted for multiplicity of testing using the Bonferroni correction, where specified.

      Results

      Cohort characteristics

      Demographics of the ILNR and ENR cohorts are summarized in Table 2. There was no significant difference in age at diagnosis, RD following primary surgical debulking, histology or grade of disease at diagnosis, or disease stage at diagnosis between the ILNR and ENR groups. These data indicate good fidelity of the ILNR–ENR matching process. Patterns of ILNR are described in Table 3.
      Table 2Demographics of isolated lymph node relapse and extranodal relapse ovarian carcinoma cohorts
      FactorClassILNR, n = 49ENR, n = 49ILNR vs ENR
      N%/rangeN%/rangeP value
      Stage at diagnosisI510.6510.21.000
      χ2 test, stage I/II vs stage III/IV
      II1021.31122.4
      III2757.42857.1
      IV510.6510.2
      NA20
      Histology at diagnosisSerous2551.03367.3.502
      χ2 test, serous/mixed vs other
      Endometrioid1224.51122.5
      Clear cell12.012.0
      Mixed histology816.348.2
      Unclassified adenocarcinoma36.100.0
      Grade at diagnosisI00.012.01.000
      χ2 test, grade I/II vs grade III
      II613.0612.2
      III4087.04275.7
      NA30
      Contemporary histologic classificationHGS3489.53186.1.733
      Fisher exact test, high-grade serous vs non-high-grade serous
      Endometrioid25.338.3
      LGS25.312.8
      Mixed HGS / endometrioid00.01
      This tumor had 2 morphologically distinct components with different immunophenotypes.
      2.8
      No specimen available1113
      Surgical debulking statusRD <2 cm3475.63370.2.733
      χ2 test, RD <2 cm vs ≥2 cm
      RD 2–5 cm715.6817.0
      RD ≥5 cm48.9612.8
      NA42
      First-line chemotherapyPlatinum2142.91734.7.693
      χ2 test
      Platinum combination2551.02857.1
      Other36.148.2
      Neoadjuvant first-line chemotherapyYes24.112.01.000
      Fisher exact test
      No4795.94898.0
      Year of diagnosis≤19992346.92142.9.667
      χ2 test
      2000–20051938.82346.9
      2006–2010714.3510.2
      Age at diagnosisMedian years6141-806241-80.339
      Welch 2-sample t test
      Specimen from diagnosisPrimary site3391.72980.6.307
      Fisher exact test, primary site vs omentum/other
      Omentum25.6616.7
      Other12.812.8
      NA20
      No specimen available1113
      ENR, extranodal relapse; ILNR, isolated lymph node relapse; NA, not available; RD, residual disease.
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      a χ2 test, stage I/II vs stage III/IV
      b χ2 test, serous/mixed vs other
      c χ2 test, grade I/II vs grade III
      d Fisher exact test, high-grade serous vs non-high-grade serous
      e χ2 test, RD <2 cm vs ≥2 cm
      f χ2 test
      g Fisher exact test
      h Welch 2-sample t test
      i Fisher exact test, primary site vs omentum/other
      This tumor had 2 morphologically distinct components with different immunophenotypes.
      Table 3Patterns of isolated lymph node relapse ovarian carcinoma
      Cases (n)Proportion of cases (%)
      ILNR pattern
       Single-site2244.9
       Multiregional
      21734.7
      3816.3
      424.1
      ILNR sites
       Para-aortic only1632.7
       Pelvic only48.2
       Inguinal only24.1
       Pelvic & para-aortic612.2
       Supraclavicular and other sites612.2
       Pelvic, para-aortic, and other(s)612.2
       Other combinations918.4
      ILNR, isolated lymph node relapse.
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.

      Clinical outcome in isolated lymph node relapse vs extranodal relapse

      ILNR patients displayed prolonged OS and PRS compared with the ENR cohort (HR = 0.55 [0.34–0.87], P = .011 and HR = 0.50 [0.31–0.80], P = .004) (Figure 1, A and Figure 1, B). The median OS and PRS in the ILNR cohort was 72.9 (95% CI 62.2–96.5) and 32.0 (95% CI 23.3–53.3) months, compared with 41.1 (95% CI 30.0–58.8) and 14.9 (95% CI 12.9–23.7) months in the ENR cohort. The length of the DFI prior to ILNR or ENR was not significantly different between the 2 cohorts (HR = 0.86 [0.60–1.29], P = .473).
      Figure thumbnail gr1
      Figure 1Clinical outcome of isolated lymph node relapse OC
      A, Overall survival in ILNR vs ENR OC. B, Postrelapse survival in ILNR vs ENR OC. C, Postrelapse survival in ILNR OC by DFI length.
      CI, confidence interval; DFI, disease-free interval; ENR, extranodal relapse; HR, hazard ratio; ILNR, isolated lymph node relapse; OC, ovarian carcinoma.
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      Multivariable analysis for OS accounting for extent of RD following primary debulking, FIGO stage, and age at diagnosis identified significantly prolonged OS in the ILNR cohort (HRmulti = 0.51 [0.31–0.84], P = .008) (Appendix: Supplementary Table S4). Multivariable analysis of PRS, accounting for DFI and age, identified prolonged PRS in ILNR cases (HRmulti = 0.52 [0.33–0.84], P = .007) (Appendix: Supplementary Table S5).
      Significantly prolonged OS (HRmulti for OS = 0.53 [0.29–0.99], P = .046) and PRS (HRmulti for PRS = 0.54 [0.31–0.96], P = .037) was demonstrated for ILNR OC when HGS cases were considered specifically (34 ILNR HGS OCs, 31 ENR HGS OCs).

      Longer disease-free interval is associated with prolonged postrelapse survival in isolated lymph node relapse ovarian carcinoma

      The importance of DFI on clinical outcome in ILNR OC remains controversial, with some authors reporting no association between DFI length and PRS or OS in this setting
      • Blanchard P.
      • Plantade A.
      • Pages C.
      • et al.
      Isolated lymph node relapse of epithelial ovarian carcinoma: outcomes and prognostic factors.
      • Ferrero A.
      • Ditto A.
      • Giorda G.
      • et al.
      Secondary cytoreductive surgery for isolated lymph node recurrence of epithelial ovarian cancer: a multicenter study.
      • Fotiou S.
      • Aliki T.
      • Petros Z.
      • et al.
      Secondary cytoreductive surgery in patients presenting with isolated nodal recurrence of epithelial ovarian cancer.
      and others reporting significant associations.
      • Legge F.
      • Petrillo M.
      • Adamo V.
      • Pisconti S.
      • Scambia G.
      • Ferrandina G.
      Epithelial ovarian cancer relapsing as isolated lymph node disease: natural history and clinical outcome.
      • Gadducci A.
      • Cosio S.
      • Zola P.
      • et al.
      The clinical outcome of epithelial ovarian cancer patients with apparently isolated lymph node recurrence: a multicenter retrospective Italian study.
      • Tu H.
      • Huang H.
      • Huang Q.D.
      • Li Z.
      • Feng Y.L.
      • Liu J.H.
      [Treatment and prognostic analysis of ovarian cancer patients with isolated region of lymph node recurrence].
      Within the ILNR cohort, DFI ≥12 months was associated with markedly prolonged PRS when accounting for patient age (HRmulti = 0.38 [0.19–0.78], P = .008), with median PRS of 47.3 months vs 20.1 months in those with DFI ≥12 months and DFI <12 months, respectively (Figure 1, C).

      Impact of isolated lymph node relapse pattern on outcome

      There was no clear differential PRS between multiregion ILNR and single-region ILNR (2 regions vs single-site HR = 1.06 [0.49–2.30], P = .890; ≥3 sites vs single-site HR = 0.94 [0.36–1.43], P = .898).
      Six ILNR cases (12.2%) involved supraclavicular LN sites. Although these cases demonstrated an apparent trend for inferior PRS (HR = 2.52 [0.95–6.69], P = .064) (Appendix: Supplementary Figure S3), there was no significant difference after accounting for DFI and age (HRmulti = 1.63 [0.58–4.60], P = .359). Other specific LN sites were not associated with apparent differential PRS (Appendix: Supplementary Table S6).

      Molecular landscape of isolated lymph node relapse high-grade serous ovarian carcinoma

      Sixty-four HGS OC cases (33 ILNR, 31 ENR) were successfully characterized for HR gene mutations and CCNE1 copy number. Frequencies of genomic abnormalities are outlined in Figure 2, A and Supplementary Table S3. Within HGS OC cases, there was no significant difference in the rate of CCNE1 copy number gain (18.2%, 6/33 vs 22.6%, 7/31, P = .900) or BRCA1/2 mutation (24.4%, 8/33 vs 19.4%, 6/31, P = .865) between the ILNR and ENR cohorts (Figure 2, A).
      Figure thumbnail gr2
      Figure 2Molecular landscape of isolated lymph node relapse and extranodal relapse ovarian carcinoma
      A, Genomic events in ILNR and ENR cases. B, Tumor-infiltrating lymphocyte burden of ILNR and ENR HGS ovarian carcinomas.
      ENR, extranodal relapse; HGS, high-grade serous; ILNR, isolated lymph node relapse; LGS, low-grade serous.
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      The CD3+ and CD8+ TIL burden was greater in diagnostic tumor specimens from HGS OC patients who went on to experience ILNR when compared with their ENR counterparts (median CD3+ cell density 1.94% vs 1.13%, P = .001 and median CD8+ cell density 0.90% vs 0.45%, P = .009; Bonferroni-adjusted P = .003 and P = .019) (Figure 2, B).

      Comment

      Principal findings

      The principal findings of this study are as follows: (1) ILNR represents a distinct pattern of OC relapse with prolonged survival vs ENR cases; (2) longer DFI prior to ILNR is associated with prolonged PRS in ILNR; (3) ILNR OC do not demonstrate significantly differential composition of known genomic subtypes associated with prognosis, namely BRCA1/2 mutation or gain of CCNE1; (4) cases that go on to experience ILNR demonstrate greater TIL burden at diagnosis compared with ENR cases.

      Study strengths and limitations

      A key strength of this study is the direct comparison of ILNR OC with matched ENR cases: a number of studies have reported ILNR as a distinct pattern of OC relapse with a relatively indolent disease course but have not systematically compared ILNR cases directly to a matched ENR cohort.
      • Blanchard P.
      • Plantade A.
      • Pages C.
      • et al.
      Isolated lymph node relapse of epithelial ovarian carcinoma: outcomes and prognostic factors.
      • Legge F.
      • Petrillo M.
      • Adamo V.
      • Pisconti S.
      • Scambia G.
      • Ferrandina G.
      Epithelial ovarian cancer relapsing as isolated lymph node disease: natural history and clinical outcome.
      • Uzan C.
      • Morice P.
      • Rey A.
      • et al.
      Outcomes after combined therapy including surgical resection in patients with epithelial ovarian cancer recurrence(s) exclusively in lymph nodes.
      • Gadducci A.
      • Cosio S.
      • Zola P.
      • et al.
      The clinical outcome of epithelial ovarian cancer patients with apparently isolated lymph node recurrence: a multicenter retrospective Italian study.
      • Tu H.
      • Huang H.
      • Huang Q.D.
      • Li Z.
      • Feng Y.L.
      • Liu J.H.
      [Treatment and prognostic analysis of ovarian cancer patients with isolated region of lymph node recurrence].
      • Ferrero A.
      • Ditto A.
      • Giorda G.
      • et al.
      Secondary cytoreductive surgery for isolated lymph node recurrence of epithelial ovarian cancer: a multicenter study.
      • Santillan A.
      • Karam A.K.
      • Li A.J.
      • et al.
      Secondary cytoreductive surgery for isolated nodal recurrence in patients with epithelial ovarian cancer.
      • Fotiou S.
      • Aliki T.
      • Petros Z.
      • et al.
      Secondary cytoreductive surgery in patients presenting with isolated nodal recurrence of epithelial ovarian cancer.
      Moreover, these studies did not perform pathology review of identified cases, precluding the ability to characterize ILNR outcome in the context of contemporary OC histotypes, which are now known to display markedly differential clinical outcome.
      • Hollis R.L.
      • Gourley C.
      Genetic and molecular changes in ovarian cancer.
      Critically, we characterize ILNR OC following contemporary histologic subtyping to facilitate investigation of ILNR in a histotype-specific manner.
      The majority of previous studies investigating ILNR have identified fewer than 20 OC cases of serous histology that go on to experience this rare relapse pattern; moreover, previous reports have not performed molecular characterization of OC cases that demonstrate ILNR.
      • Blanchard P.
      • Plantade A.
      • Pages C.
      • et al.
      Isolated lymph node relapse of epithelial ovarian carcinoma: outcomes and prognostic factors.
      • Legge F.
      • Petrillo M.
      • Adamo V.
      • Pisconti S.
      • Scambia G.
      • Ferrandina G.
      Epithelial ovarian cancer relapsing as isolated lymph node disease: natural history and clinical outcome.
      • Uzan C.
      • Morice P.
      • Rey A.
      • et al.
      Outcomes after combined therapy including surgical resection in patients with epithelial ovarian cancer recurrence(s) exclusively in lymph nodes.
      • Gadducci A.
      • Cosio S.
      • Zola P.
      • et al.
      The clinical outcome of epithelial ovarian cancer patients with apparently isolated lymph node recurrence: a multicenter retrospective Italian study.
      • Tu H.
      • Huang H.
      • Huang Q.D.
      • Li Z.
      • Feng Y.L.
      • Liu J.H.
      [Treatment and prognostic analysis of ovarian cancer patients with isolated region of lymph node recurrence].
      • Ferrero A.
      • Ditto A.
      • Giorda G.
      • et al.
      Secondary cytoreductive surgery for isolated lymph node recurrence of epithelial ovarian cancer: a multicenter study.
      • Santillan A.
      • Karam A.K.
      • Li A.J.
      • et al.
      Secondary cytoreductive surgery for isolated nodal recurrence in patients with epithelial ovarian cancer.
      • Fotiou S.
      • Aliki T.
      • Petros Z.
      • et al.
      Secondary cytoreductive surgery in patients presenting with isolated nodal recurrence of epithelial ovarian cancer.
      We identified 49 ILNR OC patients treated within the Edinburgh Cancer Centre, including 34 cases reviewed as HGS OC. This study represents the largest ILNR OC series from a single center and the only report investigating the molecular landscape of ILNR OC to date.
      Though this study does represent one of the largest reported ILNR OC cohorts, case numbers were still restricted owing to the rarity of ILNR OC. In particular, power to detect differential outcome between distinct patterns of ILNR was limited, and we could not perform meaningful analysis comparing rates of rare genomic events present in both ILNR and ENR cohorts, including mutational events in RB1, NF1, and PTEN, as well as gene-specific analysis of BRCA1 and BRCA2. Other limitations of this study include heterogeneous treatment of OC patients across the time period in which these cases were diagnosed, though diagnosis periods were comparable between the ILNR and ENR cohorts (Table 2).

      Clinical outcome in isolated lymph node relapse ovarian carcinoma

      The median PRS and OS of ILNR cases was approximately 2.7 and 6 years, consistent with previous reports of ILNR OC.
      • Blanchard P.
      • Plantade A.
      • Pages C.
      • et al.
      Isolated lymph node relapse of epithelial ovarian carcinoma: outcomes and prognostic factors.
      • Legge F.
      • Petrillo M.
      • Adamo V.
      • Pisconti S.
      • Scambia G.
      • Ferrandina G.
      Epithelial ovarian cancer relapsing as isolated lymph node disease: natural history and clinical outcome.
      • Uzan C.
      • Morice P.
      • Rey A.
      • et al.
      Outcomes after combined therapy including surgical resection in patients with epithelial ovarian cancer recurrence(s) exclusively in lymph nodes.
      • Gadducci A.
      • Cosio S.
      • Zola P.
      • et al.
      The clinical outcome of epithelial ovarian cancer patients with apparently isolated lymph node recurrence: a multicenter retrospective Italian study.
      • Tu H.
      • Huang H.
      • Huang Q.D.
      • Li Z.
      • Feng Y.L.
      • Liu J.H.
      [Treatment and prognostic analysis of ovarian cancer patients with isolated region of lymph node recurrence].
      • Ferrero A.
      • Ditto A.
      • Giorda G.
      • et al.
      Secondary cytoreductive surgery for isolated lymph node recurrence of epithelial ovarian cancer: a multicenter study.
      • Santillan A.
      • Karam A.K.
      • Li A.J.
      • et al.
      Secondary cytoreductive surgery for isolated nodal recurrence in patients with epithelial ovarian cancer.
      • Fotiou S.
      • Aliki T.
      • Petros Z.
      • et al.
      Secondary cytoreductive surgery in patients presenting with isolated nodal recurrence of epithelial ovarian cancer.
      ILNR cases displayed significantly prolonged OS and PRS compared to their ENR counterparts upon multivariable analysis (HRmulti = 0.51 and 0.52 for OS and PRS). Critically, this difference was maintained in a histotype-specific analysis of HGS cases, which account for the majority of OCs. To our knowledge, this is the first report directly demonstrating a significant difference in outcome between ILNR and ENR OC.
      Only half of the reports investigating the impact of DFI length on ILNR outcome to date have identified associations with OS or PRS.
      • Legge F.
      • Petrillo M.
      • Adamo V.
      • Pisconti S.
      • Scambia G.
      • Ferrandina G.
      Epithelial ovarian cancer relapsing as isolated lymph node disease: natural history and clinical outcome.
      • Gadducci A.
      • Cosio S.
      • Zola P.
      • et al.
      The clinical outcome of epithelial ovarian cancer patients with apparently isolated lymph node recurrence: a multicenter retrospective Italian study.
      • Tu H.
      • Huang H.
      • Huang Q.D.
      • Li Z.
      • Feng Y.L.
      • Liu J.H.
      [Treatment and prognostic analysis of ovarian cancer patients with isolated region of lymph node recurrence].
      Here, we demonstrate that DFI ≥12 months is associated with a substantial PRS benefit (median PRS approximately 3.9 vs 1.7 years), largely reflective of established associations in unselected OC cases.
      • Ushijima K.
      Treatment for recurrent ovarian cancer—at first relapse.
      Although this contradicts reports from some investigators,
      • Blanchard P.
      • Plantade A.
      • Pages C.
      • et al.
      Isolated lymph node relapse of epithelial ovarian carcinoma: outcomes and prognostic factors.
      • Ferrero A.
      • Ditto A.
      • Giorda G.
      • et al.
      Secondary cytoreductive surgery for isolated lymph node recurrence of epithelial ovarian cancer: a multicenter study.
      • Fotiou S.
      • Aliki T.
      • Petros Z.
      • et al.
      Secondary cytoreductive surgery in patients presenting with isolated nodal recurrence of epithelial ovarian cancer.
      2 of these studies reported specifically in the context of ILNR undergoing secondary debulking
      • Ferrero A.
      • Ditto A.
      • Giorda G.
      • et al.
      Secondary cytoreductive surgery for isolated lymph node recurrence of epithelial ovarian cancer: a multicenter study.
      • Fotiou S.
      • Aliki T.
      • Petros Z.
      • et al.
      Secondary cytoreductive surgery in patients presenting with isolated nodal recurrence of epithelial ovarian cancer.
      and the other compared cases using a cut-off DFI of 24 months, rather than 12 months as described here,
      • Blanchard P.
      • Plantade A.
      • Pages C.
      • et al.
      Isolated lymph node relapse of epithelial ovarian carcinoma: outcomes and prognostic factors.
      potentially explaining this discrepancy. Notably, the intervals considered in our study are akin to those used clinically to define platinum sensitivity in unselected relapsed OC.
      • Ushijima K.
      Treatment for recurrent ovarian cancer—at first relapse.
      We show no significant difference in clinical outcome between patients with ILNR at multiple sites vs those with single-site ILNR, or between distinct patterns of ILNR. Although univariable analysis suggested that supraclavicular LN involvement may confer inferior PRS, this trend was not apparent when accounting for DFI and patient age, suggesting that this is not a genuine phenomenon of supraclavicular ILNR. Notably, the number of patients with supraclavicular LN involvement was low (n = 6). Together, these data support the consideration of ILNR OC as a single disease entity, regardless of the number and location of involved sites.

      The genomic landscape of isolated lymph node relapse ovarian carcinoma

      Until now, the molecular landscape of ILNR has been completely uncharacterized. It has therefore been unclear as to whether OC cases that go on to experience ILNR demonstrate enrichment of tumors belonging to known favorable genomic subgroups. Within unselected cohorts of HGS OC, inactivation of BRCA1 or BRCA2 has been associated with favorable outcome,
      The Cancer Genome Atlas Investigators
      Integrated genomic analyses of ovarian carcinoma.
      • Tan D.S.
      • Rothermundt C.
      • Thomas K.
      • et al.
      “BRCAness” syndrome in ovarian cancer: a case-control study describing the clinical features and outcome of patients with epithelial ovarian cancer associated with BRCA1 and BRCA2 mutations.
      while copy number gain of CCNE1 has been associated with poor survival and chemoresistance.
      The Cancer Genome Atlas Investigators
      Integrated genomic analyses of ovarian carcinoma.
      • Patch A.M.
      • Christie E.L.
      • Etemadmoghadam D.
      • et al.
      Whole-genome characterization of chemoresistant ovarian cancer.
      Genomic characterization of this cohort did not identify significant depletion or enrichment of these molecular events in ILNR HGS OC cases versus their ENR counterparts. These data suggest that the survival benefit of ILNR OC is not underpinned by large-scale enrichment for BRCA1/2-mutant cases with favorable prognosis or absence of CCNE1-gained cases that have poorer prognosis, and suggest that these genomic subgroups do not display markedly differential propensity for ILNR.

      Greater tumor-infiltrating lymphocyte burden at diagnosis in patients who subsequently experience isolated lymph node relapse

      Intriguingly, assessment of the CD3+ and CD8+ cell burden in ILNR and ENR tumor material—reflective of whole T cell and cytotoxic T cell populations—uncovered significantly greater TIL burden in diagnostic tissue from patients who subsequently experienced ILNR (2-fold enrichment for CD8+ cells, approximately 1.7-fold enrichment for CD3+ cells). These data suggest that active engagement of the immune system at diagnosis impacts upon the nature of disease at relapse, and that immune-mediated control of cancer cells may contribute to the indolent disease course of ILNR OC. Indeed, these data may well be of interest in relation to the use of immune-directed therapies in cancer treatment.
      • Postow M.A.
      • Chesney J.
      • Pavlick A.C.
      • et al.
      Nivolumab and ipilimumab versus ipilimumab in untreated melanoma.
      • Muro K.
      • Chung H.C.
      • Shankaran V.
      • et al.
      Pembrolizumab for patients with PD-L1-positive advanced gastric cancer (KEYNOTE-012): a multicentre, open-label, phase 1b trial.
      However, though many ILNR cases displayed high TIL burden, some cases demonstrated relatively low levels of TILs, alluding to mechanisms beyond effective T-cell engagement at diagnosis underpinning some ILNR cases.

      Conclusion

      Collectively, the data presented here—supported by previous descriptions of apparent ILNR in the literature—demonstrate that ILNR represents a distinct pattern of OC with favorable clinical outcome when compared with ENR. Patients that go on to experience ILNR harbor greater TIL burden at diagnosis, but they do not show marked enrichment or depletion of known genomic subgroups associated with differential outcome.

      Acknowledgments

      We extend our thanks to the patients who contributed to this study and to the Edinburgh Ovarian Cancer Database, from which the clinical data reported here were retrieved. We are thankful to the Wellcome Trust Clinical Research Facility (Western General Hospital, Edinburgh, UK) for their support with the sequencing described here, and to the Nicola Murray Foundation for their generous support of the Nicola Murray Centre for Ovarian Cancer Research.

      Appendix

      Figure thumbnail fx1
      Supplementary Figure S1Isolated lymph node relapse cohort identification from the Edinburgh Ovarian Cancer Database
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      Figure thumbnail fx2
      Supplementary Figure S2Automated marker-positive cell quantification by QuPath
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      Figure thumbnail fx3
      Supplementary Figure S3Postrelapse survival of isolated lymph node relapse ovarian carcinoma with and without supraclavicular lymph node involvement
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      Supplementary Table S1Tolerances for electronic matching of extranodal relapse to isolated lymph node relapse
      FeatureILNR OC case documented diagnostic characteristicMatched ENR OC case documented diagnostic characteristicPatients
      Stage at diagnosisInadequate information to stageStage II, n = 13
      Stage IIIC, n = 1
      Stage IIBStage IIA, n = 1
      Documented histology at diagnosisAdenocarcinomaSerous papillary, n = 39
      EndometrioidMixed serous/endometrioid, n = 1
      Mixed histologySerous papillary, n = 5
      Grade at diagnosisNAGrade I, n = 13
      Grade III, n = 2
      RD following debulking<2 cmNA, n = 14
      NA2–5 cm, n = 1
      >5 cm, n = 2
      The matching criteria were relaxed for 2 fields for 2 patients and 3 fields for 1 patient, as follows: 1 x stage IIB grade III mixed histology carcinoma with RD <2 cm matched to stage IIA grade III serous carcinoma with RD <2 cm; 1 x stage IV unclassified adenocarcinoma of unknown grade and RD <2 cm matched to stage IV grade III serous carcinoma with RD <2 cm; 1 x stage IV unclassified adenocarcinoma of unknown grade and RD not available matched to stage IV grade III serous carcinoma with RD >5 cm.
      ENR, extranodal relapse; ILNR, isolated lymph node relapse; NA, not available; OC, ovarian carcinoma; RD, residual disease.
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      Supplementary Table S2Cellularity of specimens used for DNA extraction
      Tumor cellularity of macrodissected areaNumber (%) of cases
      <20%3 (4.1%)
      20–39%7 (9.5%)
      40–59%9 (12.2%)
      60–79%26 (35.1%)
      ≥80%29 (39.2%)
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      Supplementary Table S3Frequency of patients with detrimental mutations in genes sequenced on Integrated DNA Technologies gene capture high throughput sequencing panel
      GeneILNR OC with mutationENR OC with mutation
      ABCB110
      ARID1A12
      ATM20
      ATR10
      BRCA163
      BRCA233
      CTNNB111
      FANCC01
      KRAS11
      MSH211
      NF141
      PIK3CA21
      PRKDC10
      PTEN11
      RB131
      SLX401
      TP533231
      Genes with no detected mutations:

      ATRX, BAP1, BARD1, BCL2L1, BLM, BRAF, BRIP1, C11orf65, CCNE1, CDK12, CHD4, CHEK1, CHEK2, EGFR, EMSY, ERBB2, ERCC4, EZH2, FANCA, FANCB, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, GNAS, KIT, MAD2L2, MDM2, MLH1, MRE11, MSH6, MUS81, MUTYH, NBN, NDUFB2, NF2, NRAS, PALB2, PARP1, PARP2, PAXIP1, PDGFRA, PER3, PMS2, PPP2R1A, PPP2R2A, RAD50, RAD51, RAD51B, RAD51C, RAD54L, RNASEH2A, RNASEH2B, RNASEH2C, RPA1, RUNDC3B, SHFM1, SLC25A40, SLFN11, TOE1, TP53BP1, UBE2T, VRK2
      ENR, extranodal relapse; ILNR, isolated lymph node relapse; OC, ovarian carcinoma.
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      Supplementary Table S4Multivariable analysis for overall survival in isolated lymph node relapse vs extranodal relapse in ovarian carcinoma
      FactorClassNHRmultiLower 95% CIUpper 95% CIP
      Relapse typeILNR490.510.310.84.008
      ENR49RefRefRefRef
      Stage at diagnosisEarly (I/II)310.410.171.02.055
      III550.450.191.03.060
      IV10RefRefRefRef
      NA2----
      Surgical debulking statusRD <2 cm670.600.321.12.109
      RD ≥2 cm25RefRefRefRef
      NA6----
      Age at diagnosisYears1.031.001.05.050
      CI, confidence interval; ENR, extranodal relapse; HR, hazard ratio; ILNR, isolated lymph node relapse; RD, residual disease.
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      Supplementary Table S5Multivariable analysis of time to isolated lymph node relapse as a predictor of postrelapse survival in isolated lymph node relapse ovarian carcinoma
      FactorClassNHRmultiLower 95% CIUpper 95% CIP
      Relapse typeILNR490.530.330.84.007
      ENR49RefRefRefRef
      DFI≥12 months460.470.290.75.006
      <12 months52RefRefRefRef
      AgeYears1.031.011.06.006
      CI, confidence interval; DFI, disease-free interval; HR, hazard ratio.
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.
      Supplementary Table S6Univariable analyses of specific lymph node site involvement and association with postrelapse survival
      FactorClassNHRLower 95% CIUpper 95% CIP
      Supraclavicular LN involvementYes62.520.956.69.064
      No43RefRefRefRef
      Pelvic LN involvementYes200.730.351.51.393
      No29RefRefRefRef
      Inguinal LN involvementYes90.720.281.87.502
      No40RefRefRefRef
      Para-aortic LN involvementYes361.100.482.56.818
      No13RefRefRefRef
      CI, confidence interval; HR, hazard ratio; LN, lymph node.
      Hollis et al. Clinical and molecular characterization of ILNR ovarian carcinoma. Am J Obstet Gynecol 2019.

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