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Microsatellite Instability in Non-Endometrioid Ovarian Epithelial Tumors: A study of 400 cases Comparing Immunohistochemistry, PCR, and NGS Based Testing with Mutation Status of MMR Genes.
Department of Obstetrics and Gynecology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
Department of Obstetrics and Gynecology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
The Fingerland Department of Pathology, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech Republic
Department of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech RepublicDepartment of Pathology, Charles University, 3rd Faculty of Medicine, University Hospital Kralovske Vinohrady, 10034 Prague, Czech RepublicDepartment of Pathology and Molecular Medicine, Third Faculty of Medicine, Charles University, Thomayer University Hospital, Prague, Czech Republic
Department of Cellular Pathology, Barts Health NHS Trust, and Blizard Institute of Core Pathology, Queen Mary University of London, London, United Kingdom
Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
Corresponding authors: Ivana Stružinská. Department of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Studničkova 2, 12800 Prague 2, Czech Republic. Phone: +420224968658
Corresponding authors: Pavel Dundr. Department of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Studničkova 2, 12800 Prague 2, Czech Republic. Phone: +420224968624
Testing of microsatellite instability is not only used as a triage for possible Lynch syndrome, but also to predict immunotherapy treatment response. The aim of this study was to assess the frequency of mismatch repair deficiency (MMR-D) / microsatellite instability (MSI) in 400 cases of non-endometrioid ovarian tumors (high-grade serous, low-grade serous, mucinous and clear cell), to compare different methodological approaches of testing, and to assess the optimal approach for next generation sequencing (NGS) MSI testing. For all tumors, we evaluated immunohistochemical (IHC) expression of MMR proteins and assessed microsatellite markers by PCR-based method. Except for high-grade serous carcinoma, we correlated the findings of IHC and PCR with NGS-based MSI testing. We compared the results with somatic and germline mutation in MMR genes. Among the whole cohort, seven MMR-D cases, all clear cell carcinomas (CCC), were found. On PCR analysis, six cases were MSI-high and one was MSS. In all cases, mutation of an MMR gene was found; in two cases, the mutation was germline (Lynch syndrome). An additional five cases with a mutation in MMR gene(s) with MSS status and without MMR-D were identified. We further utilized sequence capture NGS for MSI testing. Employing 53 microsatellite loci provided high sensitivity and specificity. Our study shows that MSI occurs in 7% of CCC while it is rare or absent in other non-endometrioid ovarian neoplasms. Lynch syndrome was present in 2% of patients with CCC. However, some cases with MSH6 mutation can evade all testing methods, including IHC, PCR, and NGS-MSI.
Testing of colorectal and endometrial carcinomas for mismatch repair (MMR) protein expression by immunohistochemistry (IHC) or for microsatellite instability (MSI) by PCR is a well-established screening method for Lynch syndrome (LS) [
Kim SR, Tone A, Kim RH, et al. Performance characteristics of screening strategies to identify Lynch syndrome in women with ovarian cancer. Cancer-Am Cancer Soc. 2020.
Significant frequency of MSH2/MSH6 abnormality in ovarian endometrioid carcinoma supports histotype-specific Lynch syndrome screening in ovarian carcinomas.
Lynch syndrome screening in gynaecological cancers: results of an international survey with recommendations for uniform reporting terminology for mismatch repair immunohistochemistry results.
]. Some ovarian carcinomas are also associated with LS but the optimal screening strategy to identify LS in ovarian cancer patients has not been determined, yet [
Kim SR, Tone A, Kim RH, et al. Performance characteristics of screening strategies to identify Lynch syndrome in women with ovarian cancer. Cancer-Am Cancer Soc. 2020.
]. Currently, the significance of MSI testing is increasing in a broad spectrum of tumors. MMR deficiency (MMR-D) and/or presence of high MSI (MSI-H) are predictors of a favorable response to immune checkpoint inhibitor therapy in solid tumors [
Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy: Guideline From the College of American Pathologists in Collaboration With the Association for Molecular Pathology and Fight Colorectal Cancer.
]. In parallel with predictive testing in the broad spectrum of solid tumors, the knowledge about non-canonical neoplasms which can be associated with LS is increasing [
Methods used for MSI testing have been mostly validated for screening purposes in colorectal and endometrial carcinomas. However, due to technical or biological reasons there are challenges in MSI testing even in colorectal and endometrial cancer. As such, immunohistochemical testing of the mismatch repair machinery may give different results for a given germline or somatic mismatch repair gene missense mutation [
Retained mismatch repair protein expression occurs in approximately 6% of microsatellite instability-high cancers and is associated with missense mutations in mismatch repair genes.
]. However, in other tumors the value of different approaches is not clear. The three main methods for MSI testing are immunohistochemistry (IHC), PCR based approaches and NGS testing. According to the European Society for Medical Oncology (ESMO) recommendation on MSI testing for immunotherapy in cancer, the preferable method of testing in the first line is IHC with 4 antibodies (MLH1, PMS2, MSH2 and MSH6) [
ESMO recommendations on microsatellite instability testing for immunotherapy in cancer, and its relationship with PD-1/PD-L1 expression and tumour mutational burden: a systematic review-based approach.
]. PCR based approaches are for second line testing, in cases in which IHC is not evaluable or the results are inconclusive. However, these recommendations are for tumors belonging to the spectrum of cancer well-known to be associated with LS, including colorectal, endometrial, small intestine, urothelial, central nervous system and sebaceous gland neoplasms. For other tumor types, there are insufficient data to inform a recommendation. The ESMO comment on NGS MSI testing is that it has the potential to become the method of choice for all tumor types.
In contrast, a recent College of American Pathologists (CAP) guideline for testing for possible immunotherapy with immune checkpoint inhibitors, considers IHC and PCR as equal and states that one of these methods should be used for colorectal, small bowel and gastroesophageal carcinoma [
Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy: Guideline From the College of American Pathologists in Collaboration With the Association for Molecular Pathology and Fight Colorectal Cancer.
]. This guideline states that for endometrial carcinoma, IHC is favored over PCR testing while for other tumor types, the optimal method of testing has not been established, so far. NGS may be used for colorectal cancer, but a validated MSI NGS assay is needed. For small bowel, gastroesophageal and endometrial carcinoma MMR-IHC, MSI-PCR and MMR-IHC, respectively, should be preferred over NGS. NGS is currently not the method of choice for primary isolated MSI testing. However, MSI could be evaluated by NGS panel testing together with the assessment of the mutation profile of the tumor and tumor mutation burden (TMB). However, the set of microsatellite loci analyzed by NGS have been established for colorectal and endometrial carcinomas and can differ in other tumors. Therefore, the optimal NGS approach covering all solid tumors has not been assessed, yet. Despite the recommendation for IHC testing using all 4 antibodies, first-line testing for only 2 MMR proteins (PMS2 and MSH6) can be used, if required completed by second-line testing of MLH1 and MSH2 [
A recently developed rapid digital PCR technique for microsatellite instability has been successfully used for colorectal, endometrial and gastric carcinomas and also a small number of ovarian carcinomas without histological characterization. This novel technique is easy to handle and fast and turned out comparable to other PCR based techniques and immunohistochemistry [
Comparison of the Idylla MSI assay with the Promega MSI Analysis System and immunohistochemistry on formalin-fixed paraffin-embedded tissue of endometrial carcinoma: results from an international, multicenter study.
Multi-center real-world comparison of the fully automated Idylla microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer.
Idylla MSI test combined with immunohistochemistry is a valuable and cost effective strategy to search for microsatellite instable tumors of noncolorectal origin.
Kim SR, Tone A, Kim RH, et al. Performance characteristics of screening strategies to identify Lynch syndrome in women with ovarian cancer. Cancer-Am Cancer Soc. 2020.
Significant frequency of MSH2/MSH6 abnormality in ovarian endometrioid carcinoma supports histotype-specific Lynch syndrome screening in ovarian carcinomas.
]. The aim of our study was to assess the frequency of MMR-D/MSI-H in a broad spectrum of non-endometrioid ovarian tumors, to compare different methodological approaches for MSI testing, and to assess the optimal approach for NGS MSI testing using three different sets of microsatellite markers. Our sample set consisted of 400 primary ovarian epithelial tumors with serous, mucinous and clear cell morphology. For all tumors, we evaluated IHC expression of four MMR proteins and assessed the stability of microsatellite markers by the PCR-based method of five mononucleotides. Moreover, for all tumors except high-grade serous carcinoma (HGSC), we correlated the findings of IHC and PCR with NGS-based MSI testing. In all tumors in which NGS was performed, the results were also compared with mutation status in MMR genes. For tumors showing MMR-D/MSI-H status, non-neoplastic tissue was tested by NGS parallel to tumor tissue to exclude the possibility of LS.
MATERIALS AND METHODS
Samples
The archives of participating pathology departments were searched for cases diagnosed as low-grade serous carcinoma (LGSC), micropapillary subtype of serous borderline tumor (mSBT), clear cell carcinoma (CCC), mucinous carcinoma (MC) and mucinous borderline tumor (MBT). The sample set represents part of the sample set used in our previous studies [
Primary Mucinous Tumors of the Ovary: An Interobserver Reproducibility and Detailed Molecular Study Reveals Significant Overlap Between Diagnostic Categories.
]. 296 selected samples were eligible for IHC, PCR and NGS testing. These cases included 100 CCC, 75 LGSC, 29 mSBT and 92 mucinous tumors (29 MC, 49 MBT and 14 cases equivocal between MC and MBT as described in Dundr et al. 2023). Moreover, 104 tubo-ovarian high grade serous carcinomas (HGSC) were selected from the archives of the Department of Pathology, 1st Medical Faculty and General University Hospital in Prague. These cases were analyzed only by IHC and PCR methods. All LGSC, mSBT, HGSC and CCC cases were reviewed by at least two experienced pathologists and fulfilled the strict morphological and immunohistochemical criteria. The classification of mucinous tumors was based on the results of our previous study, which focused on the interobserver agreement and molecular analysis of ovarian mucinous tumors [
Primary Mucinous Tumors of the Ovary: An Interobserver Reproducibility and Detailed Molecular Study Reveals Significant Overlap Between Diagnostic Categories.
]. Clinicopathological characteristics of the analyzed tumors are in Table 1.
Table 1Patients and tumors characteristics
OCCC
LGSC
mSBT
*mucinous tumors
*HGSC
n. of samples
n= 100
n= 75
n= 29
n= 92
n= 104
age mean (range)
60.2 (34-82)
52 (19-83)
48 (25-85)
52 (17-83)
59.9 (36-81)
FIGO
I
67
11
11
84
9
II
7
3
1
1
7
III
16
42
11
5
64
IV
1
2
0
1
22
NA
9
17
6
1
2
OCCC_ ovarian clear cell carcinoma, LGSC_ low-grade serous carcinoma, mSBT – micropapillary variant of serous borderline tumor, HGSC_ high-grade serous carcinoma, FIGO classification was done according to WHO classification of female genital tumors
*mucinous tumors comprise of 29 mucinous carcinoma, 49 of mucinous borderline tumor and 14 of cases equivocal between mucinous carcinoma and mucinous borderline tumor
*HGSC cohort was tested only by MMR IHC and PCR-based method
The study has been approved by the Ethics Committee of General University Hospital in Prague in compliance with the Helsinki Declaration (No. 2140/19 S-IV). The Ethics Committee waived the requirement for informed consent, as according to the Czech Law (Act. no. 373/11, and its amendment Act no. 202/17) it is not necessary to obtain informed consent in fully anonymized studies.
Immunohistochemical expression of mismatch repair proteins
Immunohistochemistry was performed on tissue microarrays (TMAs) using 4μm thick sections of formalin-fixed and paraffin-embedded (FFPE) tissue. For construction of the TMAs, eligible areas of each tumor were identified and two tissue cores (each 2.0 mm in diameter) were taken from the donor block using the tissue microarray instrument TMA Master (3DHISTECH Ltd., Budapest, Hungary).
Immunohistochemical analysis (IHC) was performed with antibodies against MSH2 (clone FE11, ready-to-use, Dako, USA), MSH6 (clone EP49, ready-to-use, Bio SB, USA), MLH1 (clone ES05, ready-to-use, Dako, USA), and PMS2 (clone EP51, ready-tu-use, Dako, USA) using Dako Omnis (Agilent Technologies, California, USA) with the EnVision FLEX system.
Loss of MMR protein expression was defined as a loss of nuclear staining in all tumor cells for any of the four MMR proteins with preserved positive internal control (nuclear expression of endothelial cells, lymphocytes and/or stromal cells). Weak nuclear staining (weaker in comparison to the internal control) in less than 5% of tumor cells was regarded as lost. Weak nuclear staining (weaker than the internal control) in more than 5% of tumor cells or negativity of both tumor cells and internal control on the initial TMA sections were regarded as equivocal. All cases with an equivocal result on TMA, with loss of expression of any MMR protein or with an MSI-H status detected by PCR and/or NGS were immunohistochemically reanalyzed on whole tissue sections. All immunohistochemical stains were scored by at least two experienced pathologists (PD, MB, KN).
Microsatellite instability (MSI) testing by PCR and fragment analysis (PCR-FA)
A pentaplex PCR reaction was performed with fluorescent labeled primers for the set of five quasi monomorphic mononucleotide microsatellite markers BAT-26, BAT-25, NR-21, NR-22, NR-24, followed by fragmentation analysis on ABI 3500 (ThermoFisher). Size of PCR products was evaluated in GeneMapper™ Software (ThermoFisher). MSI-high (MSI-H) phenotype was defined as the presence of two or more unstable microsatellite markers. Cases with one unstable marker (MSI-low) were included in a group of MSS tumors.
Capture DNA NGS
For the purpose of this study, samples used as a part of a large project that focused on rare epithelial ovarian tumors were included. For all samples (100 CCC, 75 LGSC, 29 mSBT and 92 mucinous tumors) the NGS sequencing was part of our previous studies, the methodology is detailed there [
Primary Mucinous Tumors of the Ovary: An Interobserver Reproducibility and Detailed Molecular Study Reveals Significant Overlap Between Diagnostic Categories.
]. DNA was extracted also from the adjacent non-neoplastic tissue (Magcore Genomic DNA FFPE One step kit; RBC Bioscience) for sequencing analysis to rule out a potential germline origin of MMR gene mutation in cases with detected MMR mutation in the tumor. The NGS capture panel used in the current and previous studies included 727 genes or gene parts (2097 kbp; NimbleGen, Roche). In this study, we focused only on detected mutations in mismatch repair genes MLH1, PMS2, MSH2, MSH6, and on evaluation of microsatellite markers as described below.
MSI testing by NGS approach
The set of NGS 17 loci included standard recommended Bethesda markers BAT-26, BAT-25, NR-21, NR-22, NR-24, D5S346 [
], and microsatellite loci in cancer-related genes TGFBR2, BAX, IGF-II. These are frequently targeted by microsatellite instability and their inactivation may contribute to tumor progression [
]. In addition, MONO-27, Penta-C, ACVR2A, BTBD7, DIDO1, MRE11A, RYR3, SEC31A were included. These are used in commercial kits for testing microsatellite instability in different types of tumors, in particular, colorectal and endometrial cancer (Promega, IdyllaTM) [
Association of a novel set of 7 homopolymer indels for detection of MSI with tumor mutation burden and total indel load in endometrial and colorectal cancers.
]. The NGS raw data were processed (including trim reads, mapping reads to GrCH38 reference genome) by QIAGEN CLC Genomics Workbench software (Qiagen). Loci track of our 17 microsatellite markers were imported to the module “Detect MSI status”. Fifteen samples with optimal DNA quality and with known MMR (IHC) and MSS (PCR-FA) status were used for creating a MSI-baseline. Default setting of this module was used. The “Detect MSI Status” module measures the statistical variation of the length distribution of each microsatellite locus and determines the stability of each locus by comparing the statistical variation of the tested sample with the normal baseline samples.
To increase the specificity of MSI detection in ovarian tumors, we identified in our targeted custom panel (2097 kbp) all microsatellite markers (1–5 bp in length and comprising 5 repeats or more) by comparison with a set of genome-wide microsatellite markers described by Hause et al [
]. The algorithm for selection of ovarian-specific microsatellite loci is shown in Figure 1. Out of 10,249 microsatellite loci identified (with sufficient read depth) 36 ovarian-tumors specific loci were selected. They were unstable in at least 4 out of 6 MSI-H samples in our sample set (previously evaluated by PCR-FA), while stable in all MSS and proficient mismatch repair (MMR-P) samples. The most unstable microsatellite markers identified in >5 MSI-H tumors are summarized in Table 2.
Figure 1Examples of immunohistochemical staining for the MMR proteins.
All statistical tests were carried out using the program R (version 4.0.2, https://www.r-project.org/) and /or Statistica (TIBCO). Association between age (continuous variable) and MSI/MSS status (dichotomous variable) was evaluated using the Mann-Whitney U test. To analyze the agreement among three methodical approaches (IHC, PCR-FA and NGS), the methods of inter-rater reliability was implemented using the package “irr” in the R program (available at http://CRAN.R-project.org/package=irr). The level of agreement among three methods was described using Fleiss Kappa coefficient (κ). Consistent with prior literature on the level of agreement (Landis and Koch 1977), kappa coefficients were interpreted as poor (0.01-0.20), fair (0.21-0.40), moderate (0.41-0.60), substantial (0.61-0.80) and almost perfect (0.81-1.00). The package ‘cutpointr’ implemented in R software (https://github.com/thie1e/cutpointr) was used for the evaluation of an optimal cut-point value for sensitivity and specificity. All tests were two-sided and a p-value of less than 0.05 was considered as significant.
RESULTS
Among the whole cohort of 400 tumors, 7 MMR-D cases were found (Figure 1); 6 of these were MSI-H and one MSS on PCR analysis. All these cases were CCC. In all these tumors, mutation of an MMR gene was found. In two of these cases, the mutation was germline (MLH1 and MSH6, respectively) and in 5 cases, the mutation was somatic (in 2 cases each MSH2 and MSH6, in one case MLH1). In 5/7 cases, in which TMB was evaluable, high TMB (range 2 - 86) was found. The case with the highest TMB (TMB=86) contained a concurrent POLE mutation. In addition, there were 5 cases identified with mutation in MMR genes but with a MSS status. Two of these cases showed a germline mutation of MSH6. Two MMR proficient cases with a somatic mutation of an MMR gene (one case with MLH1 and concurrent PMS2 mutation, second case with MSH2 mutation) showed high TMB, but both showed concurrent POLE mutation. The results are summarized in Table 3.
Table 3List of cases with detected pathogenic mutation in MMR genes, including results of MMR IHC, PCR based method and NGS approach for evaluation of MMR-D/MSI status.
IHC
PCR
NGS
Mutation analysis
dg.
age at dg.
MLH1
PMS2
MSH2
MSH6
MMR-D/ MMR-P
MSI/ MSS
53 loci
MMR genes G_Germline S_Somatic
POLE gene
TMB (mut/Mb)
% of positively stained nucleus
CCC
44
98
95
0
0
MMR-D
MSI
55%
S MSH2:c.2375_2378del,p.(N792fs)
no
NA
CCC
44
1
3
95
40
MMR-D
MSI
45%
G MLH1:c.116+1G>A,p.?
no
2
CCC
39
100
70
0
45
MMR-D
MSI
66%
S MSH2:c.1386+1G>A,p.?
no
14
CCC
45
1
1
100
90
MMR-D
MSI
79%
S MLH1:c.1459C>T,p.(R487*)
no
26**
CCC
45
55
85
55
10
MMR-D
MSI
55%
S MSH6:c.1610_1613del,p.(K537fs)
no
25
CCC
59
100
100
90
5
MMR-D
MSI
55%
G MSH6:c.1238G>C,p.(W413S)
no
18**
CCC
66
100
85
90
5
MMR-D
MSS
23%
S MSH6:c.1630G>T,p.(E544*)
c.1376C>T,p.(S459F)
86
CCC
55
100
60
70
45
MMR-P
MSS
0%
S MLH1:c.1896+1G>A,p.? and S PMS2:c.1882C>T,p.(R628*)
c.1366G>C,p.(A456P)
66**
CCC
68
90
95
25
55
MMR-P
MSS
8%
S MSH6:c.1805C>G,p.(S602*)
no
7
CCC
48
100
100
100
95
MMR-P
MSS
6%
S MSH2:c.1447G>T,p.(E483*)
c.1231G>C,p.(V411L)
51
MBT
41
90
80
90
20
MMR-P
MSS
4%
G MSH6:c.2351_2352del,p.(N784fs)
no
1**
LGSC
46
80
20
90
35
MMR-P
MSS
8%
G MSH6:c.3226C>T,p.(R1076C)
no
1
dg._histological diagnosis, IHC_immunohistochemistry, MMR_miss match repair, CCC_ clear cell carcinoma, LGSC_low grade serous carcinoma, MBT_mucinous borderline tumor, PCR-FA_Polymerase chain reaction and fragment analysis, NGS_Next generation sequencing
MMR-P_proficent mismatch repair protein staining, MMR-D_deficient/loss of mismatch repair protein staining, MSI_microsatellite instable, MSS_microsatellite stable
TMB_tumor mutation burden, mut/Mb_ mutation per megabase, ** TMB in this samples could be higher because of the purity of tumors cell of the input DNA under 40%,
Thirteen cases (8 CCC, 3 LGSC, 2 mucinous tumors) were MSI-low by PCR. These tumors were classified as MSS by using the NGS approach and immunohistochemistry showed proficient expression for all MMR proteins. The most frequently detected unstable microsatellite in these cases was NR-21 (in 8 of 13).
Pathogenic mutation or likely pathogenic mutation in at least one MMR gene was found in 12/296 (4 %) cases (10 OCC, 1 MBT, 1 LGSC; Table 3). Out of those, seven cases were MMR-D, six cases were MSI-H and 6 cases were MSS. Three MSS samples with mutation in the MMR gene had a concurrent mutation in the POLE gene and had also the highest TMB (range 51-86 mut/Mb; mean 67.7). TMB in 6 MSI-H tumors ranged between 2-26 mut/Mb (mean 17 mut/Mb).
The sequence capture NGS-based approach for MSI testing showed higher sensitivity and specificity using 53 selected microsatellite loci, based on the analysis of our MMR-D and MSS cases compared to using 17 microsatellite loci typical for colorectal and endometrial carcinoma and to analyzing all microsatellite loci present in our NGS DNA panel (Figure 2). Using the 17 microsatellite loci panel, the MMR-D cases showed between 18-65% of unstable microsatellites (mean 30%). The cut-off value for MSI-H status by this panel was 18% of unstable loci (sensitivity=1, specificity= 0.97, AUC = 0.996). Using the 53 microsatellite loci panel the MMR-D cases showed 23-79% of unstable microsatellites (mean 51%). The optimal cut-off value for MSI-H status by this panel was 23% of unstable loci (sensitivity=1, specificity=1, AUC= 1). Analysis of all microsatellite loci (10,249) showed a cut-off value of 5% for MSI-H (sensitivity = 1, specificity = 0.65, AUC = 0.807).
Figure 2Design of NGS-based approach for the testing of microsatellite instability in ovarian tumors.
PCR-FA_polymerase chain reaction and fragment analysis (5 mononucleotide markers); IHC MMR_ immunohistochemical analysis of 4 mismatch repair protein MLH1, MSH2, MSH6, and PMS2; NGS – next generation sequencing (capture DNA NGS, panel of 727 genes including MLH1, MSH2, MSH6 and PMS2); MSI_microsatellite instability; MMR-D_deficient mismatch repair; MMR-P_proficient mismatch repair; MSS-baseline was calculated from randomly chosen set of MSS samples with sufficient read depth for automated analysis in CLC Genomic Workbench software (Qiagen).
As the best result of both specificity and sensitivity was reached by the 53-loci panel, this panel was selected for further analysis of methods agreement.
The agreement between evaluation of MMR/MSI by different methods (IHC/PCR-FA/NGS – 53 loci panel) was almost perfect (Kappa = 0.949, Z = 27.9, p < 0.001). Only one case showed discrepancy in the PCR approach (MSS) versus NGS and IHC (MSI in both methods).
Clinicopathological correlations
The average age of MMR-D CCC patient was significantly lower compared to MSS CCC patients (mean/median age 48.9/45 years versus 61.5/62 years, p < 0.05). All seven MMR-D cases were FIGO stage I.
There was no significant difference for the onset of the disease between patients with germline (n = 4) and somatic (n = 8) MMR gene mutations (age 47.5 and 51.2, respectively, p = 0.610).
DISCUSSION
It has been shown that 15-25% of all ovarian carcinomas are related to hereditary factors, of which 10-15% are associated with LS [
]. For patients with LS, the estimated lifetime risk of developing ovarian carcinoma is between 6-12% and up to 38% (10-38%) for those with germline MSH2 mutation [
Kim SR, Tone A, Kim RH, et al. Performance characteristics of screening strategies to identify Lynch syndrome in women with ovarian cancer. Cancer-Am Cancer Soc. 2020.
]. Several studies have shown that the ovarian tumor types associated with LS are mostly endometrioid carcinoma followed by CCC, the latter accounting for about 12-14% of ovarian carcinomas associated with LS [
Kim SR, Tone A, Kim RH, et al. Performance characteristics of screening strategies to identify Lynch syndrome in women with ovarian cancer. Cancer-Am Cancer Soc. 2020.
]. In HGSC, germline pathogenic variants have been described in approximately 25% of cases, mainly in genes involved in the DNA damage response pathway such as BRCA1, BRCA2, BRIP1, PALB2, RAD51, ATM, and CHEK2 [
According to the literature, the frequency of MMR-D/MSI in ovarian endometrioid carcinomas is lower compared to endometrioid endometrial carcinomas and occurs in about 12% of cases (range 0-22.9%) [
Kim SR, Tone A, Kim RH, et al. Performance characteristics of screening strategies to identify Lynch syndrome in women with ovarian cancer. Cancer-Am Cancer Soc. 2020.
Significant frequency of MSH2/MSH6 abnormality in ovarian endometrioid carcinoma supports histotype-specific Lynch syndrome screening in ovarian carcinomas.
]. The published literature concerning the frequency of MSI in non-endometrioid ovarian tumors is equivocal. In low-grade and high-grade serous carcinomas, the frequency of MSI is absent to very low with a range of 0-4.2% (average 0.3%) [
Significant frequency of MSH2/MSH6 abnormality in ovarian endometrioid carcinoma supports histotype-specific Lynch syndrome screening in ovarian carcinomas.
Comprehensive analysis of PD-L1 expression, HER2 amplification, ALK/EML4 fusion, and mismatch repair deficiency as putative predictive and prognostic factors in ovarian carcinoma.
Mismatch repair deficiency is associated with MSI phenotype, increased tumor-infiltrating lymphocytes and PD-L1 expression in immune cells in ovarian cancer.
Relationship between Microsatellite Instability, Immune Cells Infiltration, and Expression of Immune Checkpoint Molecules in Ovarian Carcinoma: Immunotherapeutic Strategies for the Future.
]. However, the results could be potentially influenced by using a PCR based method with 2 mononucleotides and 3 dinucleotide microsatellites. We think that using dinucleotide microsatellites could overestimate MSI in ovarian tumors, as it has been shown that a panel of five mononucleotide repeats is more suitable [
]. Our results support the low frequency of MSI in ovarian serous tumors, low-grade and high-grade, as in our cohort of 208 tumors all were MSS. Only two other studies analyzed SBT (together 70 cases), all of them showing MSS, which is in concordance with the results of our study [
Significant frequency of MSH2/MSH6 abnormality in ovarian endometrioid carcinoma supports histotype-specific Lynch syndrome screening in ovarian carcinomas.
Comprehensive analysis of PD-L1 expression, HER2 amplification, ALK/EML4 fusion, and mismatch repair deficiency as putative predictive and prognostic factors in ovarian carcinoma.
Mismatch repair deficiency is associated with MSI phenotype, increased tumor-infiltrating lymphocytes and PD-L1 expression in immune cells in ovarian cancer.
Relationship between Microsatellite Instability, Immune Cells Infiltration, and Expression of Immune Checkpoint Molecules in Ovarian Carcinoma: Immunotherapeutic Strategies for the Future.
]. One might speculate that in the Segev study also seromucinous tumors were included which are related to endometrioid tumors and were not studied by us. Our results support the finding that MSI is rare in ovarian MC, as all our mucinous tumors were MSS. For CCC, the published literature shows an MSI frequency of 0-25% (average 5.4%), which concurs with our finding of 7% [
Kim SR, Tone A, Kim RH, et al. Performance characteristics of screening strategies to identify Lynch syndrome in women with ovarian cancer. Cancer-Am Cancer Soc. 2020.
Significant frequency of MSH2/MSH6 abnormality in ovarian endometrioid carcinoma supports histotype-specific Lynch syndrome screening in ovarian carcinomas.
Diffuse Intratumoral Stromal Inflammation in Ovarian Clear Cell Carcinoma is Associated With Loss of Mismatch Repair Protein and High PD-L1 Expression.
Altogether, we detected 12 patients with class 4/5 mutation of any MMR genes, including 10 patients with CCC, one with LGSC and one with MBT. Mutations were somatic in 8 and germline in 4 patients, including two CCC, one LGSC and one MBT. In three patients with somatic MMR gene mutation (two MSS, one MMR-D) a concurrent POLE mutation was found. All these mutations in MMR genes were classified as pathogenic or likely pathogenic (class 4/5) with the exception of one case with the germline MSH6 variant NM_000179.2:c.1238G>C, p.(Trp413Ser). This variant is assessed as a variant of uncertain significance, according to the ClinVar database. However, our case with this variant was MMR-D/MSI-H, and along with the findings of one previous study, which detected this variant in one colorectal and one endometrial carcinoma associated with LS, this variant should be considered likely pathogenic (class 4) [
Our study showed a 100% specificity of MSI testing for the detection of MMR gene mutation. The sensitivity was rather low, as we found MMR-D/MSI-H status in only 7/12 (58%) cases. The 5 discordant cases included a somatic mutation of MLH1 and concurrent PMS2 in one case and of MSH2 in the second case. Both occurred in POLE mutant tumors. All remaining discordant cases were MSH6 mutated, two of them (LGSC and MBT) with germline mutation. Our results are similar to others showing that MSI testing can be false negative in up to half of MSH6-mutated cases and could lead to false-negative screening tests for some LS patients with a MSH6 mutation [
]. The only discrepant case between IHC and PCR testing in our study was a case with a somatic MSH6 mutation showing MMR-D, but a false negative result on PCR based testing.
It has been demonstrated that an NGS approach for MSI testing can be used as an alternative to IHC or PCR based methods, but one should be aware that the spectrum of microsatellite loci can differ among tumor types and different loci can be preferentially mutated in different tumors [
]. All previous studies focusing on unstable microsatellite loci in different tumor types used data from The Cancer Genome Atlas (TCGA). Some of these studies focused on the description of unstable loci occurring in stomach, colorectal and endometrial carcinomas, but ovarian carcinomas were not included [
]. One study analyzed 32 different tumor types. Although the results were promising, they failed to identify a set of unstable microsatellite loci with sufficient sensitivity and specificity, which could be used in ovarian and breast carcinoma [
]. These results further suggested that microsatellite loci are tumor specific and tissue-specific loci should be used for optimal NGS MSI testing, which was confirmed by our study. Comparing three sets of microsatellites, our study showed that 17 microsatellites typical for colorectal carcinoma and microsatellites selected based on the results of our MSI testing by IHC and PCR) have high sensitivity and specificity and could be used in routine practice. However, we are aware of the limitations of our study. The definition of an optimal microsatellite set for ovarian carcinomas would require validation on a confirmatory sample set, but that was not the goal of this study.
In conclusion, the results of our study show that MSI occurs in 7% of ovarian CCC. Absence of MSI in other tumor types suggests that occurrence of MSI in these tumors is unusual, which is concordant with the majority of the published literature. In our unselected population of CCC, 2% of patients had LS. Our results confirm that in addition to ovarian EC, also cases of ovarian CCC should be routinely tested for MSI as a part of LS screening and to identify candidate patients for immunotherapy. IHC testing of MMR protein expression seems to be superior to PCR based testing. NGS-MSI could be used in practice, but validation of microsatellite sets optimal for this testing in ovarian tumors is needed. Finally, our results suggest that germline mutation of MMR genes and possibly LS can rarely occur in ovarian tumors other than CCC and endometrioid carcinoma. However, some of these cases, particularly, with MSH6 mutation would be missed by all testing methods, including IHC, PCR, and NGS-MSI.
DATA AVAILABILITY
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
AUTHORS CONTRIBUTION
PD performed study concept and design. NH collected and interpreted data and prepared manuscript, IS prepared the manuscript. All authors participated on material preparation, data collection and / or data analyses. RM provided statistical analysis. All authors read and approved the final paper.
Brief commentary
Background
The significance of microsatellite testing in ovarian tumours is increasing. Testing is not only used as a triage for possible Lynch syndrome, but also to predict immunotherapy treatment response. But the optimal screening strategy to identify ovarian carcinomas patients associated with Lynch syndrome or microsatellite instable ovarian tumours has not been determined, yet.
Translational Significance
Precision assessing of the frequency of mismatch repair deficiency and/or microsatellite instability in non-endometrioid ovarian tumours. Comparison of different methodological approaches of testing and assessing of the optimal approach for next generation sequencing (NGS).
Declaration of Competing Interest
The authors have no conflicts of interest that are relevant to the content of this article to declare. All authors have read the journal's authorship agreement.
ACKNOWLWDGEMENTS
This work was supported by the Ministry of Health, Czech Republic (MH CZ DRO-VFN 64165 and AZV NV19-03-00007), by Charles University (Project UNCE204065), and by the European Regional Development Fund (EF16_013/0001674 and BBMRI.cz reg. no. LM2023033).
The authors wish to thank Mgr. Zachary Harold Kane Kendall, B.A. (Institute for History of Medicine and Foreign Languages, First Faculty of Medicine, Charles University) for the English proofreading.
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Diffuse Intratumoral Stromal Inflammation in Ovarian Clear Cell Carcinoma is Associated With Loss of Mismatch Repair Protein and High PD-L1 Expression.
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