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Lynch syndrome-associated epithelial ovarian cancer and its immunological profile

Open AccessPublished:July 15, 2021DOI:https://doi.org/10.1016/j.ygyno.2021.07.001

      Highlights

      • Lynch syndrome-associated ovarian cancers show activation of immune response and immune evasion mechanisms.
      • High levels of CD3 and CD8 positive lymphocytes were seen in 60% and 53% of the tumors, respectively.
      • Loss of B2M was observed in 47% of the cancers.
      • PD-L1 expression was found in 28% of the tumors.
      • Mismatch repair status correlated with microsatellite instability status in 60% of the cancers.

      Abstract

      Introduction

      Lynch syndrome is a multi-tumor syndrome characterized by mismatch repair deficiency (MMR-d), microsatellite instability (MSI), and increased tumor-infiltrating lymphocytes (TILs) making these tumors candidates for treatment with immune checkpoint inhibitors. However, response may depend on tumor-induced immune evasion mechanisms, e.g. loss of Beta-2-Microglobulin (B2M) or upregulation of programmed death protein ligand 1 (PD-L1). We investigated the immune response and B2M and PD-L1 expression in Lynch syndrome-associated ovarian cancers.

      Methods

      We successfully analyzed 30 Lynch syndrome-associated epithelial ovarian cancers collected through the Danish Hereditary Non-Polyposis Colorectal Cancer (HNPCC) register. MMR-d, MSI, immune response (CD3, CD8, and CD68), and immune evasion mechanisms (B2M and PD-L1) were investigated. Statistical associations between these markers were evaluated in addition to survival in relation to B2M/PD-L1.

      Results

      Of the 29 evaluable tumors, 27 were MMR-d (93.1%). Likewise of 26 evaluable tumors, 14 were MSI (53.8%). MMR-d/MMR-proficiency associated with MSI/MSS in 60.0%. Half of the ovarian tumors presented with high levels of TILs. Loss of B2M expression was observed in 46.7% of the tumors, while expression of PD-L1 was seen in 28.0% of the cases. There was no association between B2M/PD-L1 and MSI/TILs/survival. Loss of B2M was often seen in tumors with low TILs (p = 0.056 or p = 0.059 for CD3 and CD8 positive cells, respectively).

      Conclusion

      MMR-d, MSI, and TILs are also seen in Lynch syndrome-associated ovarian cancers making these potential candidates for checkpoint-based immunotherapy. The clinical impact from immune evasion through loss of B2M needs to be investigated further in larger cohorts.

      Keywords

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      To our knowledge, B2M or PD-L1 expression has not been studied in Lynch syndrome-associated ovarian cancers. With the long-term aim to investigate the potential benefit of immune checkpoint therapy in Lynch syndrome ovarian cancer, we analyzed B2M and PD-L1 expression and correlated it to the host-induced immune response and MSI status.

      1. Materials and methods

      1.1 Patient cohort

      All women with verified pathogenic germline variants in the MMR genes MLH1, MSH2, MSH6 and PMS2 and their first-degree relatives diagnosed with ovarian cancer between January 1st, 1980 to September 24th, 2019 were identified through the Danish Hereditary Non-Polyposis Colorectal Cancer (HNPCC) Register (N = 83). In here, 60 epithelial ovarian tumors were surgically removed and available from 55 women belonging to 52 Lynch syndrome families. In total, 35 cancer specimens were collected, from which sufficient formalin-fixed paraffin-embedded tumor tissues were obtained from 30 cases. Of the 30 cases, 27 were carriers of germline variants (17 MSH2, 8 MSH6, 1 MLH1, and 1 PMS2), while 3 were first-degree relatives to carriers (2 having a first-degree relative with an MSH6 variant and 1 with a first-degree relative with an MSH2 variant).
      Clinical data such as MMR gene variant, family relation, age at diagnosis, and surgery date were extracted from the HNPCC register, while tumor stage, differentiation grade, and morphology were supplemented with data from pathology reports. When mixed morphology was seen the tumors were classified according to the predominant morphology. Ethical approval for the study was obtained by the Scientific and Ethics Committee of the Capital Region of Copenhagen, Denmark (H-17001916) and reviewed by the Data Protection Agency (AHH-2017-071).

      1.2 Immunohistochemical staining

      Immunohistochemical staining was performed at Institute of Clinical Sciences, Division of Oncology and Pathology, Lund University for MLH1 (clone ES05, PT link high pH, 1:50, Agilent, California, USA), MSH2 (clone FE11, PT link high pH, 1:50, Agilent, California, USA), MSH6 (clone EP49, PT link high pH, 1:50, Agilent, California, USA), PMS2 (clone EP51, PT link high pH, 1:40, Agilent, California, USA), and B2M (polyclonal A0072, no pretreatment, 1:600, Agilent, California, USA) using a Dako Autostainer Plus (Agilent, California, USA) and the Envision™ FLEX or Envision™ FLEX+ kit (Agilent, California, USA) according to manufacturer's instructions [
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      Immunoprofiles of Colorectal Cancer from Lynch syndrome.
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      Department of Pathology, Amager and Hvidovre Hospital performed immunohistochemistry for CD3 (polyclonal GA503, PT link low pH, Agilent, California, USA), CD8 (clone C8/144B, PT link high pH, Agilent, California, USA), CD68 (clone KP1, PT link high pH, Agilent, California, USA), and PD-L1 (clone 22C3, PT link low pH, Agilent, California, USA) using the DAKO OMNIS (Agilent, California, USA) and the Envision™ FLEX or Envision™ FLEX+ kit (Agilent, California, USA).
      Whole slides of 3 μM were used for B2M and PD-L1, while tissue microarray (containing 2 × 1 mm biopsies from tumor areas located using Hematoxylin and Eosin whole slide stainings from each case) was used for CD3, CD8, CD68, MLH1, MSH2, MSH6, and PMS2. Control tissues such as brain, placenta, normal epithelial ovarian tissue, and tonsils were used where appropriate. A pilot study of 15 tumors investigating the correlation between whole slide and tissue microarray scorings for CD3 and CD8, respectively, found a high correlation between the two different methods (Spearman's correlation ranged from 0.889 to 0.956).

      1.3 Immunohistochemical evaluation of mismatch repair deficiency

      MMR-d was investigated in all 30 specimens with 1 case being non-evaluable (MSH6 germline variant carrier). Stainings were scored positive if >10% of the tumor cells showed loss of protein expression for MLH1, PMS2, and MSH2 and >20% for MSH6. All immunohistochemical stainings were evaluated independently by two observers (MR and CT) and discussed whenever discordance occurred. If concordance was not reached a third observer (MV – mentioned in acknowledgements) was included. If the stainings could not be scored based on TMA sections, whole slides were used (N = 2). Of the three genetically untested individuals, two individuals had MMR-d tumors and they were therefore assumed to be induced by underlying germline MMR gene variants. The last untested first-degree relative presented with an MMR-proficient tumor with MSS status. Although this tumor likely was induced sporadically, it was included to increase variation during statistical analyses. However, it was not included in the survival analysis since individuals with survival less than a month from surgery were excluded. In 1 of the 26 germline carriers, no evidence of MMR-d was observed, although this individual had a pathogenic MSH2 variant.

      1.4 Immunohistochemical evaluation of tumor infiltrating immune cells

      Whenever possible, 10 high power fields (HPF) of tumor areas were scored for each tumor specimen. Tumor areas were defined as having between 80 and 100% tumor cells/HPF. HPFs were defined by one microscopic view using 40× magnification objectives with a Leica DM LB microscope.
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      ]. Given the lack of standardized cutoffs, we counted the exact number of TILs and macrophages. When discriminating between low and high infiltration, we defined ≥20 infiltrating immune cells as high infiltration [
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      ]. The exact number of tumor-infiltrating immune cells were counted by two observers (MR and CT) with a Spearman's correlation of 0.971–0.993 for the mean values of the 10 HPF scored by each observer. For the results, the mean value from the two observers was used.

      1.5 Immunohistochemical evaluation of B2M and PD-L1

      For B2M immunohistochemical evaluation, the protein expression was evaluated on whole slides and was deemed lost when >50% of the tumor cells showed loss of protein expression. B2M expression was scored by two independent observers (MR and CT) and discussed to reach concordance. For two cases, reduced B2M expression were observed and characterized as retained B2M expression.
      Immunohistochemical PD-L1 expression was evaluated on whole slides for 26 of the samples with sufficient tissue. The expression was reported by the combined positive score (CPS) defined as the number of PD-L1 staining cells (viable tumor cells, and lymphocytes and macrophages within tumor nests or next to it) divided by viable tumor cells x 100. PD-L1 expression was defined as CPS ≥1 [
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      1.6 Microsatellite instability analysis

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      ]. A new Hematoxylin and Eosin section was made from each tumor block and evaluated by a pathologist (KS or MF) to mark areas with at least 5% tumor. One tumor was excluded due to lack of tumor tissue and three were excluded because there was too little tissue left in the block. The analysis was performed according to the manufacturer's instructions (Biocartis, Belgium) with the exception that the chosen tumor area was macro dissected from the sample with a thickness of 10 μM, and for small biopsies two macro dissections were used.

      1.7 Statistical analyses

      The inter-observer variabilities were analyzed using Spearman's correlation for the continuous variables (presented above).
      For correlation between categorical variables, Fishers exact test was used while Spearman's non-parametric rank correlation was used to find the correlation between numerical variables. The non-parametric Kruskal-Wallis test was used for associations between categorical and numeric variables.
      Five-year crude survival following surgery for ovarian cancer was visualized using the Kaplan-Meier model (for all with survival longer than a month from surgery) and estimated using multivariate Cox proportional hazard regression adjusting for age at epithelial ovarian cancer diagnosis and tumor stage.
      All the analyses were performed in R version 3.6.0 [
      • Core Team R.
      R: A Language and Environment for Statistical Computing.
      ] and significance was considered reached at p < 0.05.

      2. Results

      In total, 30 epithelial ovarian cancers from Lynch syndrome families were analyzed. The ovarian cancers developed in carriers or first-degree relatives of carriers with genetic variations in MLH1 (n = 1), MSH2 (n = 18), MSH6 (n = 10), and PMS2 (n = 1) with a mean age of 47.3 years (Table 1). Stage I or II were seen in 89.3% of the tumors with only three being stage III or IV and 55.6% of the cases were endometrioid.
      Table 1Clinical data, mismatch repair deficiency, and microsatellite instability data in Lynch syndrome-associated epithelial ovarian cancers.
      CharacteristicsOvarian cancers (N = 30)
      Age (range and mean)47.3 (30–69)
      Carrier status N (%)N = 30
       Carrier27 (90%)
       First-degree3 (10%)
      Genes N (%)N = 30
      MLH11 (3.3%)
      MSH218 (60.0%)
      MSH610 (33.3%)
      PMS21 (3.3%)
      Stage N (%)N = 28
       I-II25 (89.3%)
       III-IV3 (10.7%)
      Morphology N (%)N = 27
       Endometrioid15 (55.6%)
       Clear cell6 (22.2%)
       Mucinous3 (11.1%)
       Serous3 (11.1%)
      Differentiation N (%)N = 22
       High-grade
      Medium high to high-grade differentiation.
      5 (22.7%)
       Low-grade
      Low to medium high-grade differentiation.
      17 (77.3%)
      MMR-deficiency
      In accordance to underlying germline MMR variant.
      N (%)
      N = 29
       Yes27 (93.1%)
       No2 (6.9%)
      Microsatellite instability N (%)N = 26
       Yes14 (53.8%)
       No12 (46.2%)
      a Medium high to high-grade differentiation.
      b Low to medium high-grade differentiation.
      c In accordance to underlying germline MMR variant.

      2.1 MMR-deficiency/MSI

      Immunohistochemical analyses showed loss of MMR protein expression in accordance to the underlying germline MMR mutation for 27 out of 29 cases (93.1%) (Table 1). Sufficient MMR and MSI data were obtained from 25 tumors in which the MSI assay predicted 15 cases according to their MMR status (60%). Out of the 15 tumors, 14 was both MMR-d and MSI while one case was both MMR-proficient and MSS. The 10 discrepant tumors were MMR-d and MSS.

      2.2 Tumor-infiltrating immune cells

      The intratumoral immune response profile showed high levels (≥20 cells) of infiltration of CD3, CD8, and CD68 positive cells in 60.0%, 53.3%, and 46.7% of the tumors, respectively (Fig. 1). As expected, high levels of CD3 infiltrative levels were associated with high levels of CD8 infiltrative cells with a Pearson's correlation coefficient of 0.938 (p < 0.001). The correlation between CD3 and CD68 was 0.526 (p = 0.003) while it was 0.482 (p = 0.007) for CD8 and CD68. The level of tumor-infiltrating immune cells was not associated with an MSI phenotype (p = 0.269 for CD3, p = 0.425 for CD8, p = 0.797 for CD68).
      Fig. 1
      Fig. 1Immunohistochemical staining with different levels of tumor-infiltrating CD3 (A), CD8 (C), and CD68 (E) positive cells. Boxplots showing the distribution of tumors with increasing levels of infiltration of immune cells (0–19, 20–49, 50–99 and above 100) for CD3 (B), CD8 (D), and CD68 (F) positive cells.

      2.3 Immune evasion mechanisms

      Immune evasion through loss of B2M protein expression was observed in 14 out of 30 tumors (46.7%) (Fig. 2B ).
      Fig. 2
      Fig. 2Immunohistochemistry of B2M expression (A) and loss of B2M expression (B). Boxplot showing the correlation between B2M loss and mean tumor-infiltrating CD3 positive cells (C) and mean tumor-infiltrating CD8 positive cells (D).
      Loss of B2M expression was not associated with levels of tumor infiltrating CD3 positive cells (p = 0.056), CD8 positive cells (p = 0.059), or CD68 positive cells (p = 0.662), although there seemed to be a trend for lower numbers of CD3/CD8 positive cells when B2M was lost (Fig. 2C and D). There was no significant correlation between MSI and B2M protein expression (p = 1).
      PD-L1 expression was successfully analyzed for 25 tumors and was positive in 28.0%. There was no association between the levels of TILs and PD-L1 protein expression (p = 0.128 for CD3 and p = 0.133 for CD8) (Fig. 3C and D ) or between PD-L1 and B2M protein expression (p = 0.378). Likewise, there was no association between PD-L1 and MSI (p = 1).
      Fig. 3
      Fig. 3Immunohistochemistry of PD-L1 expression (A) and loss of PD-L1 expression (B). The association between PD-L1 expression and mean CD3 positive lymphocytes (C) or mean CD8 positive lymphocytes (D).
      B2M loss or PD-L1 expression were not associated with survival (p = 0.500 and p = 0.899, respectively) (Fig. 4).
      Fig. 4
      Fig. 45-year crude survival in relation to B2M expression (A) and PD-L1 expression (B). No significant difference was observed in either case.

      3. Discussion

      Immune checkpoint therapy has shown a great potential during the latest years with the PD-1 inhibitor pembrolizumab as the first tissue-agnostic drug to achieve FDA approval in 2017 based on a 39.6% response rate in MSI solid tumors [
      • Lemery S.
      • Keegan P.
      • Pazdur R.
      First FDA approval agnostic of Cancer site - when a biomarker defines the indication.
      ]. Motivated by improved survival rates and complete response rates of 18.6%, multiple clinical trials have been initiated to investigate immune checkpoint inhibitors for treatment of MSI/MMR-d cancers, including ovarian cancers [
      • Lemery S.
      • Keegan P.
      • Pazdur R.
      First FDA approval agnostic of Cancer site - when a biomarker defines the indication.
      ,
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      • Piha-Paul S.A.
      • Doi T.
      • Gao B.
      • Chung H.C.
      • Lopez-Martin J.
      • Bang Y.J.
      • Frommer R.S.
      • Shah M.
      • Ghori R.
      • Joe A.K.
      • Pruitt S.K.
      • Diaz L.A.
      Efficacy of pembrolizumab in patients with noncolorectal high microsatellite instability/mismatch repair-deficient cancer: results from the phase II KEYNOTE-158 study.
      ]. To explore the immunotherapeutic potential in Lynch syndrome ovarian cancer, we analyzed the immune response and immune evasion mechanisms in epithelial ovarian cancer associated with Lynch syndrome and found high levels of TILs in more than half of the tumors, loss of B2M in 46.7%, and PD-L1 expression in 28.0%.
      MMR-d induces multiple frameshift mutations, particularly in repetitive DNA regions causing MSI, which in turn are presented via the HLA class I receptors as neoantigens that recruit and activate the immune system [
      • Kloor M.
      • von Knebel Doeberitz M.
      • Gebert J.F.
      Molecular testing for Microsatellite Instability and its value in tumor characterization.
      ]. The high levels of TILs observed in our ovarian cancer cohort, indicate that half of these Lynch syndrome MMR-d tumors, like other Lynch syndrome cancers, e.g. of the endometrium and colorectum, are highly immunogenic and may activate the immune system via neoantigen presentation [
      • Smyrk T.C.
      • Watson P.
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      Distinct immunological landscapes characterize inherited and sporadic mismatch repair deficient endometrial cancer.
      ]. These tumors have been suggested to evade the immune system through protein loss of the HLA class I subunit B2M, which is likely achieved by mutations in the microsatellite regions in the coding regions of the B2M gene [
      • Kloor M.
      • Michel S.
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      • Büttner R.
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      • Benner A.
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      Beta2-microglobulin mutations in Microsatellite unstable Colorectal tumors.
      ,
      • Walkowska J.
      • Kallemose T.
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      • Langkilde A.
      • Nilbert M.
      • Therkildsen C.
      Immunoprofiles of Colorectal Cancer from Lynch syndrome.
      ,
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      ]. In our cohort, loss of B2M expression was found in 47.6% of the cases, which is higher than previous frequencies of 19–30% reported for MMR-d colorectal cancer [
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      ]. Loss of B2M may inhibit tumor recognition by cytotoxic CD8 positive T cells, leaving these tumors resistant to immune checkpoint inhibitors as observed in malignant melanoma patients [
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      • Kloor M.
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      • Benner A.
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      • Kuchenbuch B.
      • von Knebel Doeberitz M.
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      ,
      • Echterdiek F.
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      • Benner A.
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      ]. Here, B2M loss was seen in 58.3% and 57.1% of the tumors with low CD3 and CD8 positive TILs, respectively. However, as in previous studies in Lynch syndrome colorectal cancers, no statistical association was found in this study [
      • Walkowska J.
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      • Andersen O.
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      • Langkilde A.
      • Nilbert M.
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      • Echterdiek F.
      • Janikovits J.
      • Staffa L.
      • Müller M.
      • Lahrmann B.
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      • Hartog B.
      • Nelius N.
      • Benner A.
      • Tariverdian M.
      • von Knebel Doeberitz M.
      • Grabe N.
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      Low density of FOXP3-positive T cells in Normal colonic mucosa is related to the presence of Beta2-microglobulin mutations in Lynch syndrome-associated Colorectal Cancer.
      ]. The co-existence of loss of B2M expression and low TILs could be explained by the sequence of events occurring during tumor development with high levels of activated lymphocytes present in normal tissue and early precursor lesions, which may mediate clonal loss of B2M expression during tumor progression, in turn leading to less infiltration [
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      ]. Alternatively, an explanation for a lack of association could be found in the hypermutable microsatellite region in the B2M gene, which is likely to be affected by the underlying germline MMR-d [
      • Clendenning M.
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      Somatic mutations of the coding microsatellites within the Beta-2-microglobulin gene in mismatch repair-deficient colorectal cancers and adenomas.
      ].
      Immune evasion may also be achieved through upregulation of PD-L1, which may inactivate the cytotoxic T cells and dampen the antitumor activity [
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      ]. Activation of the PD-1/PD-L1 signaling pathway has been observed in peripheral tissues and is suspected to play an essential role in inhibition of T cell-mediated immune response and protection of the normal tissue from immune-mediated tissue damage [
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      ]. Despite a variety of antibodies, differences in cut-off points and evaluation of PD-L1 expression, PD-L1 remains a heavily studied predictive biomarker for response to immunotherapy [
      • Arora S.
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      ]. In advanced ovarian cancer, PD-L1 expression has been correlated with response to pembrolizumab treatment and a higher response was seen in tumors with a higher PD-L1 expression [
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      ]. In this Lynch syndrome cohort, we found PD-L1 expression in 28.0% of the tumors, which is lower than that reported for sporadic ovarian cancers (46–51%) also when using the same scoring criteria and antibody as in this study [
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      ]. The lower percentage of PD-L1 expression may be explained by the characteristics of our Lynch syndrome cohort, including early stage and endometrioid subtype, since PD-L1 expression has been associated with advanced stages and aggressive histologic subtypes in epithelial ovarian cancer [
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      ]. Another hypothesis could be that immune evasion through B2M loss may render additional immune editing unnecessary since B2M loss and PD-L1 expression were rarely observed in the same tumor (2 out of 25). Although PD-L1 expression in theory may hold a promising predictive value, we suggest broader molecular analyses for several biomarkers, including MSI and B2M, to aid in the selection of possible responders to immunotherapy.
      MSI and MMR-d are interchangeably used as the first pan-cancer biomarkers for prediction of response to anti-PD-1/PD-L1-therapy [
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      PD-1 blockade in tumors with mismatch-repair deficiency.
      ] but these biomarkers may not be in complete concordance when focusing on Lynch syndrome-associated cancers. Although MSI has been seen in 98% of the MMR-d colorectal cancers [
      • Loughrey M.B.
      • McGrath J.
      • Coleman H.G.
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      • Maxwell P.
      • McGready C.
      • Bingham V.
      • Humphries M.P.
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      • James J.A.
      Identifying mismatch repair-deficient colon cancer: near-perfect concordance between immunohistochemistry and microsatellite instability testing in a large, population-based series.
      ] this is not the case for Lynch syndrome-associated gynecological cancers. When focusing on MMR-d tumors, 61–85% of these are MSI-H for endometrial cancers and 39% for ovarian cancer [
      • Crosbie E.J.
      • Ryan N.A.J.
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      • Green K.
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      Assessment of Mismatch Repair deficiency in ovarian Cancer.
      ,
      • Ryan N.A.J.
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      • Crosbie E.J.
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      ,
      • Stelloo E.
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      • Church D.N.
      • Morreau H.
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      ]. Our ovarian cancer data add to this observation with an agreement between the two assays of only 56%. Here, we used the Idylla™ assay to investigate the MSI status, which may affect the concordance between the two assays. However, when compared with Promega MSI, Idylla™ has a 100% concordance, specificity, and sensitivity for both colorectal cancer and endometrial cancer [
      • Gilson P.
      • Levy J.
      • Rouyer M.
      • Demange J.
      • Husson M.
      • Bonnet C.
      • Salleron J.
      • Leroux A.
      • Merlin J.-L.
      • Harlé A.
      Evaluation of 3 molecular-based assays for Microsatellite Instability detection in formalin-fixed tissues of patients with endometrial and Colorectal cancers.
      ]. In addition, the Promega assay did not show a higher concordance with MMR-d than Idylla™ presented in here when analyzed in the ovarian cancer study by Crosbie et al. [
      • Crosbie E.J.
      • Ryan N.A.J.
      • McVey R.J.
      • Lalloo F.
      • Bowers N.
      • Green K.
      • Woodward E.R.
      • Clancy T.
      • Bolton J.
      • Wallace A.J.
      • McMahon R.F.
      • Evans D.G.
      Assessment of Mismatch Repair deficiency in ovarian Cancer.
      ]. The different concordance between MSI and MMR-d in colorectal and gynecological cancers may reflect alternative tumorigenic development across tissue types. Hence, MSI analyses may not be the correct approach to identify all tumor types that may be candidates for immunotherapy and broader molecular analyses, e.g. tumor mutation burden, may improve the prediction of response [
      • Arora S.
      • Velichinskii R.
      • Lesh R.W.
      • Ali U.
      • Kubiak M.
      • Bansal P.
      • Borghaei H.
      • Edelman M.J.
      • Boumber Y.
      Existing and emerging biomarkers for immune checkpoint immunotherapy in solid tumors.
      ].
      The rarity of Lynch syndrome and particularly Lynch syndrome-associated ovarian cancers limits our study cohort. We aimed to include 60 cases over a period of 40 years, but only reached 35 cases of which 5 had insufficient material. Although no oncology data are present in the national HNPCC register, immunotherapy could be ruled out simply due to the age of specimens (all being surgically removed before November 2017). The number of included biomarkers and their variability and overlap of expression pattern may weaken the possible predictive value of each of the biomarkers, warranting larger studies with clinical outcome to fully understand the complexity and response patterns in Lynch syndrome ovarian cancer. As the focus of this paper was Lynch syndrome, we did not include sporadic cancers although it could be valuable as a comparative cohort, as these are diagnosed in more advanced stages. Our study primarily included stage I and II cancers, which are unlikely to be offered immunotherapy. However, as immunotherapy is currently tested on a broad spectrum of different MSI tumor types, including ovarian cancers, and as some Lynch syndrome ovarian epithelial cancers are diagnosed at advanced stages (and with recurrence), the immune response and evasion mechanisms are relevant to explore in both sporadic and Lynch syndrome cohorts.

      4. Conclusion

      This is to our knowledge one of the first Lynch syndrome ovarian cancer cohorts in which MSI status, immune infiltration, PD-L1 expression, and B2M expression are evaluated. Lynch syndrome-associated ovarian cancer show MSI and are infiltrated with CD3 and CD8 positive lymphocytes and may thus be potential candidates for checkpoint-based immunotherapy. Although these data need validation in larger cohorts with clinical outcome, we hypothesize that tumors with high levels of TILs and normal expression of B2M may be more likely to respond to immunotherapy, while B2M negative and poorly infiltrated tumors may be resistant to therapy. The complexity of the immune response and tumor evasion mechanisms need to be investigated with other platforms as well as in larger studies.

      Author contributions

      Maria Rasmussen: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Writing - original draft, Visualization, and Funding acquisition. Kevin Lim: Resources, Investigation, and Writing - original draft. Eva Rambech: Resources. Mads Hald Andersen: Conceptualization and Writing - review and editing. Inge Marie Svane: Conceptualization and Writing - review and editing. Lars Henrik Jensen: Conceptualization and Writing - review and editing. Ove Andersen: Writing - review and editing, Supervision, and Project administration. Mef Nilbert: Conceptualization, Validation, Writing - review and editing, Supervision, Project administration, and Funding acquisition. Christina Therkildsen: Conceptualization, Methodology, Investigation, Writing - review and editing, Supervision, Project administration, and Funding acquisition.

      Declaration of Competing Interest

      The authors have no conflicts of interests to declare.

      Acknowledgements

      We would like to acknowledge the Department of Pathology at Amager and Hvidovre Hospital, Hvidovre, for their helpful discussions, their help with the analyses and evaluation of the immunohistochemical stainings, especially Jesper Hansen Bonde, Mogens Vyberg, Luc Phuc Dong, Helle Pedersen, Kåre Simonsen, and Maria Franzmann.
      We would also like to acknowledge Mats Jönsson at the Department of Pathology and Oncology, Lund University, for the B2M analyses. In addition, we would like to acknowledge Thomas Kallemose at Department of Clinical Research at Amager and Hvidovre Hospital, Hvidovre, for statistical inputs.
      We would also like to acknowledge all the Danish pathology departments for lending us the tumor tissues and all the Danish clinical genetic and surgical departments for identifying and reporting the families to the Danish HNPCC Register and thereby helping to maintain an updated register.
      Financial support was granted from the Danish Cancer Society Research Center ( A12599 and A14570 ) and from the Amager and Hvidovre Hospital research funds ( 2019 and 2020 ).

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