Immunohistochemical mismatch repair deficiency versus PCR microsatellite instability: a tale of two methodologies in endometrial carcinomas

Endometrial carcinomas are common gynecological malignancies. Microsatellite instability and mismatch repair deficiency may be detected in endometrial carcinomas and tumours associated with Lynch syndrome. We aimed to compare results obtained using immunohistochemical mismatch repair (MMR) antibodies and polymerase chain reaction (PCR) for microsatellite instability (MSI) on endometrioid endometrial carcinomas (EEC) at a South African state hospital. Once ethical clearance was obtained, 145 cases of EEC were retrieved. These cases were subjected to immunohistochemistry (IHC) for MLH1, PMS2, MSH6 and MSH2 antibodies; and multiplex MSI PCR for the markers BAT-25, BAT-26, NR-27, NR-24, and NR-21. Cases demonstrating MMR and MSI discordance and cases showing loss of MLH1 staining then underwent MLH1 promoter methylation testing. Mismatch repair deficiency was noted in 28.28% of 145 cases, whilst 37.1% showed MSI by PCR. The overall accuracy was 69.29%. There were 37 cases showing loss of MLH1 staining and MMR/MSI discordance was detected in 25 cases. These cases underwent hypermethylation assessment which was identified in 72.13% of cases. The current study shows that 25 (17.24%) out of 145 cases would not have had abnormalities identified if PCR MSI had not been performed and would not have been flagged as having a possible germline mutation. Most (68%) of these 25 cases were hypermethylated. We therefore recommend that endometrial carcinomas undergo both screening tests in South Africa for patients under the age of 70 years. Tissue specimens may be tested for MSH6 and PMS2 immunohistochemical stains in addition to PCR MSI testing.

Endometrial carcinomas; Immunohistochemical mismatch repair deficiency; PCR microsatellite instability.

[1] Peterson L.M., Kipp B.R., Halling K.C., Kerr S.E., Smith D.I., Dis-tad T.J., et al.: “Molecular characterization of endometrial cancer”. Int. J. Gynecol. Pathol., 2012, 31, 195-205.

[2] Garcia-Dios D.A., Lambrechts D., Coenegrachts L., Vandenput I., Capoen A., Webb P.M., et al.: “High-throughput interrogation of PIK3CA, PTEN, KRAS, FBXW7 and TP53 mutations in primary endometrial carcinoma”. Gynecol. Oncol., 2013, 128, 327-334.

[3] South African National Cancer Registry. National Cancer Registry: “Cancer in South Africa 2016”. Johannesburg: Accessed at https://www.nicd.ac.za/wp-content/uploads/2020/04/NCR_2016_ Report_updated_14April2020.pdf [2016, July 2, 2020].

[4] Arabi H., Guan H., Kumar S., Cote M., Bandyopadhyay S., Bryant C., et al.: “Impact of microsatellite instability (MSI) on survival in high grade endometrial carcinoma”. Gynecol. Oncol., 2009, 113, 153- 158.

[5] Levine A.J., Phipps A.I., Baron J.A., Buchanan D.D., Ahnen D.J., Cohen S.A., et al.: “Clinicopathologic risk factor distributions for MLH1 promoter region methylation in CIMP-positive tumors”. Cancer Epidemiol. Biomarkers Prev., 2016, 25, 68-75.

[6] Matias-Guiu X., Prat J.: “Molecular pathology of endometrial carcinoma”. Histopathology, 2013, 62, 111-123.

[7] Yeramian A., Moreno-Bueno G., Dolcet X., Catasus L., Abal M., Colas E., et al.: “Endometrial carcinoma: molecular alterations involved in tumor development and progression”. Oncogene, 2013, 32, 403-413.

[8] Wadee R., Grayson W.: “A potpourri of pathogenetic pathways in endometrial carcinoma with a focus on Lynch Syndrome”. Ann. Diagn. Pathol., 2019, 39, 92-104.

[9] Wadee R., Grayson W.: “Identification of possible Lynch syndrome in endometrial carcinomas at a public hospital in South Africa”. Southern African Journal of Gynaecological Oncology, 2020, 12, 6- 12.

[10] Mills A.M., Liou S., Ford J.M., Berek J.S., Pai R.K., Longacre T.A.: “Lynch syndrome screening should be considered for all patients with newly diagnosed endometrial cancer”. Am. J. Surg. Pathol., 2014, 38, 1501-1509.

[11] Lax S.F., Kendall B., Tashiro H., Slebos R.J.C., Ellenson L.H.: “The frequency of p53, kras mutations, and microsatellite instability differs in uterine endometrioid and serous carcinoma”. Cancer, 2000, 88, 814-824.

[12] Lax S.F.: “Molecular genetic changes in epithelial, stromal and mixed neoplasms of the endometrium”. Pathology (Phila.), 2007, 39, 46-54.

[13] Risinger J.I., Maxwell G.L., Chandramouli G.V.R., Aprelikova O., Litzi T., Umar A., et al.: “Gene expression profiling of microsatellite unstable and microsatellite stable endometrial cancers indicates distinct pathways of aberrant signaling”. Cancer Res., 2005, 65, 5031-5037.

[14] Kanopienė D., Smailytė G., Vidugirienė J., Bacher J.: “Impact of microsatellite instability on survival of endometrial cancer patients”. Medicina (Mex.), 2014, 50, 216-221.

[15] Karamurzin Y., Rutgers J.K.L.: “DNA mismatch repair deficiency in endometrial carcinoma”. Int. J. Gynecol. Pathol., 2009, 28, 239-255.

[16] Lynch H., Lynch P., Lanspa S., Snyder C., Lynch J., Boland C.: “Review of the Lynch syndrome: history, molecular genetics, screening, differential diagnosis, and medicolegal ramifications”. Clin. Genet., 2009, 76, 1-18.

[17] Stewart A.: “Genetic testing strategies in newly diagnosed endometrial cancer patients aimed at reducing morbidity or mortality from lynch syndrome in the index case or her relatives”. PLoS Curr., 2013, 5, ecurrents. eogt. b59a6e84f27c536e50db4e46aa26309c.

[18] Liccardo R., De Rosa M., Izzo P., Duraturo F.: “Novel implications in molecular diagnosis of lynch syndrome”. Gastroenterol. Res. Pract., 2017, 2017, 2595098.

[19] Chao E.C.: “Molecular models for the tissue specificity of DNA mismatch repair-deficient carcinogenesis”. Nucleic Acids Res., 2006, 34, 840-852.

[20] Kato A., Sato N., Sugawara T., Takahashi K., Kito M., Makino K., et al.: “Isolated loss of PMS2 immunohistochemical expression is frequently caused by heterogenous MLH1 promoter hypermethyla-tion in lynch syndrome screening for endometrial cancer patients”. Am. J. Surg. Pathol., 2016, 40, 770-776.

[21] Peltomäki P.: “Epigenetic mechanisms in the pathogenesis of Lynch syndrome”. Clin. Genet., 2014, 85, 403-412.

[22] Cho K.R., Cooper K., Croce S., Djordevic B., Herrington S., Howitt B., et al.: “International society of gynecological pathologists (IS-GYP) endometrial cancer project”. Int. J. Gynecol. Pathol., 2019, 38, S114-S122.

[23] Shia J., Ellis N.A., Klimstra D.S.: “The utility of immunohisto-chemical detection of DNA mismatch repair gene proteins”. Vir-chows Arch., 2004, 445, 431-441.

[24] Hampel H., Frankel W., Panescu J., Lockman J., Sotamaa K., Fix D., et al.: “Screening for Lynch syndrome (hereditary nonpolyposis colorectal cancer) among endometrial cancer patients”. Cancer Res., 2006, 66, 7810-7817.

[25] Tafe L.J., Riggs E.R., Tsongalis G.J.: “Lynch syndrome presenting as endometrial cancer”. Clin. Chem., 2014, 60, 111-121.

[26] Zhang L.: “Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome”. J. Mol. Diagn., 2008, 10, 301-307.

[27] Hayden M.J., Nguyen T.M., Waterman A., Chalmers K.J.: “Multiplex-Ready PCR: A new method for multiplexed SSR and SNP genotyping”. BMC Genomics, 2008, 9, 80.

[28] Wang Y., Shi C., Eisenberg R., Vnencak-Jones C.L.: “Differences in microsatellite instability profiles between endometrioid and colorectal cancers”. J. Mol. Diagn., 2017, 19, 57-64.

[29] McConechy M.K., Talhouk A., Li-Chang H.H., Leung S., Huntsman D.G., Gilks C.B., et al.: “Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability (MSI) phenotype in endometrial carcinomas”. Gynecol. Oncol., 2015, 137, 306-310.

[30] Meyer L.A., Broaddus R.R., Lu K.H.: “Endometrial cancer and lynch syndrome: clinical and pathologic considerations”. Cancer Control, 2009, 16, 14-22.

[31] Haghighi M.M., Javadi G.R., Parivar K., Milanizadeh S., Zali N., Fatemi S.R., et al.: “Frequent MSI mononucleotide markers for diagnosis of hereditary nonpolyposis colorectal cancer”. Asian Pac. J. Cancer Prev., 2010, 11, 1033-1035.

[32] Ehrich M., Nelson M.R., Stanssens P., Zabeau M., Liloglou T., Xi-narianos G., et al.: “Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry”. Proc. Natl. Acad. Sci., 2005, 102(44), 15785-90.

[33] Pérez-Carbonell L., Alenda C., Payá A., Castillejo A., Barberá V. M., Guillén C., et al.: “Methylation analysis of MLH1 improves the selection of patients for genetic testing in lynch syndrome”. J. Mol. Diag., 2010, 12, 498-504.

[34] Olkhov-Mitsel E., Bapat B.: “Strategies for discovery and validation of methylated and hydroxymethylated DNA biomarkers”. Cancer Medicine, 2012, 1, 237-260.

[35] Watson P.F., Petrie A.: “Method agreement analysis: A review of correct methodology”. Theriogenology, 2010, 73, 1167-1179.

[36] Suchiman H.E.D., Slieker R.C., Kremer D., Slagboom P.E., Heijmans B.T., Tobi E.W.: “Design, measurement and processing of region-specific DNA methylation assays: the mass spectrometry-based method EpiTYPER”. Front. Genet., 2015, 6, 287.

[37] Goldstein J.B., Wu W., Borras E., Masand G., Cuddy A., Mork M. E., et al.: “Can microsatellite status of colorectal cancer be reliably assessed after neoadjuvant therapy?” Clin. Cancer Res., 2017, 23, 5246-5254.

[38] Chaves P., Cruz C., Lage P., Claro I., Cravo M., Leitão C.N., et al.: “Immunohistochemical detection of mismatch repair gene proteins as a useful tool for the identification of colorectal carcinoma with the mutator phenotype”. J. Pathol., 2000, 191, 355-360.

[39] Stelloo E., Jansen A.M.L., Osse E.M., Nout R.A., Creutzberg C.L., Ruano D., et al.: “Practical guidance for mismatch repair-deficiency testing in endometrial cancer”. Ann. Oncol., 2017, 28, 96-102.

[40] Joost P., Veurink N., Holck S., Klarskov L., Bojesen A., Harbo M., et al.: “Heterogenous mismatch-repair status in colorectal cancer”. Diagn. Pathol., 2014, 9, 126.

[41] Sari A., Pollett A., Eiriksson L.R., Lumsden-Johanson B., Van de Laar E., Kazerouni H., et al.: “Interobserver agreement for mismatch repair protein immunohistochemistry in endometrial and nonserous, nonmucinous ovarian carcinomas”. Am. J. Surg. Pathol., 2019, 43, 591-600.

[42] Remo A., Fassan M., Lanza G.: “Immunohistochemical eval-uation of mismatch repair proteins in colorectal carcinoma”. AIFEG/GIPAD proposal., 2016, 108, 104-109.

[43] Nuovo G.: “False-positive results in diagnostic immunohistochem-istry are related to horseradish peroxidase conjugates in commercially available assays”. Ann. Diagn. Pathol., 2016, 25, 54-59.

[44] Chiaravalli A.M., Furlan D., Facco C., Tibiletti M.G., Dionigi A., Casati B., et al.: “Immunohistochemical pattern of hMSH2/hMLH1 in familial and sporadic colorectal, gastric, endometrial and ovarian carcinomas with instability in microsatellite sequences”. Virchows Arch., 2001, 438, 39-48.

[45] Gown A.M.: “Diagnostic immunohistochemistry”. Arch. Pathol. Lab. Med., 2016, 140, 893-898.

[46] Nordic Immunohistochemical Quality Control, MLH1 run 49 2017 [Internet]. 2017 [cited 2018 Nov 19]. Available from: http://www. nordiqc.org/downloads/assessments/86_81.pdf

[47] Nordic Immunohistochemical Quality Control, PMS2 run 53 2018 [Internet]. 2018 [cited 2018 Nov 19]. Available from: http://www. nordiqc.org/downloads/assessments/104_84.pdf

[48] Nordic Immunohistochemical Quality Control, MSH2 run 50 2017. 2018.

[49] Nordic Immunohistochemical Quality Control, MSH6 run 52 2018 [Internet]. 2018 [cited 2018 Nov 19]. Available from: http://www. nordiqc.org/downloads/assessments/101_83.pdf

[50] Hashmi A.A., Ali R., Hussain Z.F., Faridi N., Khan E.Y., Bakar S. M.A., et al.: “Mismatch repair deficiency screening in colorectal carcinoma by a four-antibody immunohistochemical panel in Pakistani population and its correlation with histopathological parameters”. World J. Surg. Oncol., 2017, 15, 116.

[51] Niu B.T., Hammond R.F.L., Leen S.L.S., Faruqi A.Z., Trevisan G., Gilks C.B., et al.: “Artefactual punctate MLH1 staining can lead to erroneous reporting of isolated PMS2 loss”. Histopathology, 2018, 73, 703-705.

[52] de Leeuw W.J.F., Dierssen J., Vasen H.F.A., Wijnen J.T., Kenter G. G., Meijers-Heijboer H., et al.: “Prediction of a mismatch repair gene defect by microsatellite instability and immunohistochemical analysis in endometrial tumours from HNPCC patients”. J. Pathol., 2000, 192, 328-335.

[53] Dierssen J.W.F.: “Molecular pathology of mismatch repair deficient tumours with emphasis on immune escape mechanisms”. Doctoral thesis, Leiden University 2010.

[54] Zighelboim I., Powell M.A., Babb S.A., Whelan A.J., Schmidt A.P., Clendenning M., et al.: “Epitope-positive truncating MLH1 mutation and loss of PMS2: implications for IHC-directed genetic testing for lynch syndrome”. Familial Cancer, 2009, 8, 501-504.

[55] Watson N., Grieu F., Morris M., Harvey J., Stewart C., Schofield L., et al.: “Heterogeneous staining for mismatch repair proteins during population-based prescreening for hereditary nonpolyposis colorectal cancer”. J. Mol. Diagn., 2007, 9, 472-478.

[56] Watkins J.C., Nucci M.R., Ritterhouse L.L., Howitt B.E., Sholl L. M.: “Unusual mismatch repair immunohistochemical patterns in endometrial carcinoma”. Am. J. Surg. Pathol., 2016, 40, 909-916.

[57] Wing-Cheuk Wong R., Palicelli A., Hoang L., Singh N.: “Interpretation of p16, p53 and mismatch repair protein immunohistochemistry in gynaecological neoplasia”. Diagn. Histopathol., 2020, 26, 257- 277.

[58] Cicek M.S., Lindor N.M., Gallinger S., Bapat B., Hopper J.L., Jenkins M.A., et al.: “Quality assessment and correlation of microsatellite instability and immunohistochemical markers among population- and clinic-based colorectal tumors”. J. Mol. Diagncs., 2011, 13, 271-281.

[59] Pastrello C., Baglioni S., Tibiletti M.G., Papi L., Fornasarig M., Morabito A., et al.: “Stability of BAT26 in tumours of hereditary nonpolyposis colorectal cancer patients with MSH2 intragenic deletion”. Eur. J. Hum. Genet., 2006, 14, 63-68.

[60] Bruegl A., Djordjevic B., Urbauer D., Westin S., Soliman P., Lu K., et al.: “Utility of MLH1 methylation analysis in the clinical evaluation of Lynch syndrome in women with endometrial cancer”. Curr. Pharm. Des., 2014, 20, 1655-1663.

[61] Shia J., Holck S., DePetris G., Greenson J.K., Klimstra D.S.: “Lynch syndrome-associated neoplasms: a discussion on histopathology and immunohistochemistry”. Familial Cancer, 2013, 12, 241-260.

[62] Esteller M.: “Cancer epigenomics: DNA methylomes and histone-modification maps”. Nat. Rev. Genet., 2007, 8, 286-298.

[63] Salvesen H.B., MacDonald N., Ryan A., Iversen O.E., Jacobs I.J., Akslen L.A., et al.: “Methylation of hMLH1 in a population-based series of endometrial carcinomas”. Clin. Cancer Res., 2000, 6, 3607-3613.

[64] Resnick K., Straughn J.M., Backes F., Hampel H., Matthews K.S., Cohn D.E.: “Lynch syndrome screening strategies among newly diagnosed endometrial cancer patients”. Obstet. Gynecol., 2009, 114, 530- 536.

[65] Garg K., Soslow R.A.: “Lynch syndrome (hereditary non-polyposis colorectal cancer) and endometrial carcinoma”. J. Clin. Pathol., 2009, 62, 679-684.

[66] Kunitomi H., Banno K., Yanokura M., Takeda T., Iijima M., Nakamura K., et al.: “New use of microsatellite instability analysis in endometrial cancer”. Oncol. Lett., 2017, 14, 3297-3301.

[67] Marcus L., Lemery S.J., Keegan P., Pazdur R.: “FDA approval summary: pembrolizumab for the treatment of microsatellite instability-high solid tumors”. Clin. Cancer Res., 2019, 25, 3753-3758.