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Küçük Hücreli Dışı Akciğer Karsinomlarında EGFR Mutasyon Analizinde Real-Time PCR ve Mutasyon Özgü İmmünohistokimya Karşılaştırması

Year 2024, Volume: 6 Issue: 2, 117 - 125, 30.06.2024
https://doi.org/10.52827/hititmedj.1351295

Abstract

Amaç: Bu çalışmanın amacı, Küçük Hücreli Dışı Akciğer Kanseri (KHDAK) hastalarında EGFR genindeki aktivasyon mutasyonlarını tespit etmek ve EGFR-tirozin kinaz inhibitörlerinin (TKI) tedavi yanıtlarıyla olan ilişkisini değerlendirmektir. Küçük hücreli dışı akciğer kanseri (KHDAK) hastalarında epidermal büyüme faktörü reseptörü (EGFR) genindeki aktive edici mutasyonların belirlenmesi, EGFR-tirozin kinaz inhibitörleri (TKI) tedavi yanıtlarıyla ilişkilidir. Mutasyon spesifik antikorlar kullanılarak yapılan immünohistokimya, belirli mutant EGFR proteinlerini tespit edebilmektedir. KHDAK hastalarında EGFR mutasyon durumunu tespit etmek için Real-Time PCR ve immünohistokimyayı karşılaştırdık ve immünohistokimyanın duyarlılık, özgüllük, pozitif ve negatif öngörü değerlerini analiz ettik.
Gereç ve Yöntem: RT- PCR ile EGFR mutasyon durumu için analiz edilen 788 küçük hücreli dışı akciğer kanseri örneğini değerlendirildi. Bu hastalar arasında 126 EGFR mutasyonlu vakayı tespit edildi. 47 EGFR mutasyonlu hastanın histolojik materyali ve sitolojik örneklerin hücre bloklarına yönelik ekzon 19 delesyonları (15 bp E746-A750) ve ekzon 21 nokta mutasyonu (L858R) için mutasyon spesifik EGFR immünohistokimyası (IHK) çalışıldı ve sonuçlar boyama kuvveti ve yaygınlığına göre değerlendirildi.
Bulgular: 47 vakadan 32’si (%68) ekzon 19 delesyonuna sahipti, bunların 14’ünde (%30) ekzon 21’de nokta
mutasyonu vardı ve birinde (%2) ekzon 18 mutasyonu gözlendi. EGFR ekzon 19 (15 bp E746-A750 delesyon) antikoru %100 duyarlılık, %40 özgüllük, %100 negatif öngörü değeri ve %78 pozitif öngörü değeri gösterdi. Ekzon 21 (L858R nokta mutasyonu) antikorunun duyarlılığı %93, özgüllüğü %91, negatif öngörü değeri %97 ve pozitif öngörü değeri %82 idi.
Sonuç: Araştırmamız, EGFR immünohistokimyasının, ekzon 21 mutasyonu için belirgin bir duyarlılık ve
özgüllük sergilediğini göstermektedir. Ancak, duyarlı olan ekzon 19 (15 bp E746-A750 delesyon) antikoru
özgüllükten yoksundur. Pozitif immünohistokimya sonucu, hastada EGFR mutasyonu olabileceğini ön görebilir ve hasta potansiyel olarak TKI tedavisine uygun olabilir; ancak bu durum tek başına belirleyici olmamalıdır. İmmünohistokimya sonuçları negatifse, doğru tanı ve uygun tedavi rehberliği için moleküler testlere başvurulması esastır.

Ethical Statement

Bu çalışma Hacettepe Üniversitesi Girişimel Olmayan Klinik Araştırmalar Etik Kurulu'ndan çalışma için etik izni alındıktan sonra (karar no: Go 13/519-24), Hacettepe Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi tarafından 1146 numaralı araştırma projesi olarak desteklendi.

Supporting Institution

Hacettepe Üniversitesi

Project Number

1146

References

  • Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021;71:209-249.
  • Jakobsen E, Olsen KE, Bliddal M, et al. Forecasting lung cancer incidence, mortality, and prevalence to year 2030. BMC Cancer 2021;21:985.
  • George J, Lim JS, Jang SJ, et al. Comprehensive genomic profiles of small cell lung cancer. Nature 2015;524:47-53.
  • Ruiz-Cordero R, Devine WP. Targeted Therapy and Checkpoint Immunotherapy in Lung Cancer. Surg Pathol Clin 2020;13:17-33.
  • Alexander M, Kim SY, Cheng H. Update 2020: Management of Non-Small Cell Lung Cancer. Lung 2020;198:897-907.
  • Liu X, Wang P, Zhang C, Ma Z. Epidermal growth factor receptor (EGFR): A rising star in the era of precision medicine of lung cancer. Oncotarget 2017;8:50209-50220.
  • Yu J, Kane S, Wu J, et al. Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clin Cancer Res 2009;15:3023-3028.
  • Kitamura A, Hosoda W, Sasaki E, et al. Immunohistochemical detection of EGFR mutation using mutation-specific antibodies in lung cancer. Clin Cancer Res 2010;16:3349-3355.
  • Rossi G, Ragazzi M, Tamagnini I, et al. Does Immunohistochemistry Represent a Robust Alternative Technique in Determining Drugable Predictive Gene Alterations in Non-Small Cell Lung Cancer? Curr Drug Targets 2017;18:13-26.
  • Kozu Y, Tsuta K, Kohno T, et al. The usefulness of mutation-specific antibodies in detecting epidermal growth factor receptor mutations and in predicting response to tyrosine kinase inhibitor therapy in lung adenocarcinoma. Lung Cancer 2011;73:45-50.
  • Simonetti S, Molina MA, Queralt C, et al. Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer. J Transl Med 2010;8:135.
  • Seo AN, Park TI, Jin Y, et al. Novel EGFR mutation-specific antibodies for lung adenocarcinoma: highly specific but not sensitive detection of an E746_A750 deletion in exon 19 and an L858R mutation in exon 21 by immunohistochemistry. Lung Cancer 2014;83:316-323.
  • Prenzel N, Fischer OM, Streit S, et al. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr Relat Cancer 2001;8:11-31.
  • Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129-2139.
  • Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497-1500.
  • Cancer Genome Atlas Research N. Comprehensive molecular profiling of lung adenocarcinoma. Nature 2014;511:543-550.
  • Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947-957.
  • Ettinger DS, Aisner DL, Wood DE, et al. NCCN Guidelines Insights: Non-Small Cell Lung Cancer, Version 5.2018. J Natl Compr Canc Netw 2018;16:807-821.
  • Warth A, Penzel R, Brandt R, et al. Optimized algorithm for Sanger sequencing-based EGFR mutation analyses in NSCLC biopsies. Virchows Arch 2012;460:407-414.
  • Brevet M, Arcila M, Ladanyi M. Assessment of EGFR mutation status in lung adenocarcinoma by immunohistochemistry using antibodies specific to the two major forms of mutant EGFR. J Mol Diagn 2010;12:169-176.
  • Houang M, Sioson L, Clarkson A, et al. EGFR mutation specific immunohistochemistry is a useful adjunct which helps to identify false negative mutation testing in lung cancer. Pathology 2014;46:501-508.
  • Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005;97:339-346.
  • Allo G, Bandarchi B, Yanagawa N, et al. Epidermal growth factor receptor mutation-specific immunohistochemical antibodies in lung adenocarcinoma. Histopathology 2014;64:826-839.
  • Leighl NB, Rekhtman N, Biermann WA, et al. Molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors: American Society of Clinical Oncology endorsement of the College of American Pathologists/International Association for the study of lung cancer/association for molecular pathology guideline. J Clin Oncol 2014;32:3673-3679.
  • Cooper WA, Yu B, Yip PY, et al. EGFR mutant-specific immunohistochemistry has high specificity and sensitivity for detecting targeted activating EGFR mutations in lung adenocarcinoma. J Clin Pathol 2013;66:744-748.
  • Remon J, Majem M. EGFR mutation heterogeneity and mixed response to EGFR tyrosine kinase inhibitors of non small cell lung cancer: a clue to overcoming resistance. Transl Lung Cancer Res 2013;2:445-448.
  • Jakobsen JN, Sorensen JB. Intratumor heterogeneity and chemotherapy-induced changes in EGFR status in non-small cell lung cancer. Cancer Chemother Pharmacol 2012;69:289-299.
  • Fassina A, Corradin M, Zardo D, et al. Role and accuracy of rapid on-site evaluation of CT-guided fine needle aspiration cytology of lung nodules. Cytopathology 2011;22:306-312.
  • Lindeman NI, Cagle PT, Aisner DL, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Mol Diagn 2018;20:129-159.
  • Hasanovic A, Ang D, Moreira AL, Zakowski MF. Use of mutation specific antibodies to detect EGFR status in small biopsy and cytology specimens of lung adenocarcinoma. Lung Cancer 2012;77:299-305.
  • Uruga H, Mino-Kenudson M. ALK (D5F3) CDx: an immunohistochemistry assay to identify ALK-positive NSCLC patients. Pharmgenomics Pers Med 2018;11:147-155.
  • Luk PP, Selinger CI, Mahar A, Cooper WA. Biomarkers for ALK and ROS1 in Lung Cancer: Immunohistochemistry and Fluorescent In Situ Hybridization. Arch Pathol Lab Med 2018;142:922-928.
  • Loo E, Khalili P, Beuhler K, et al. BRAF V600E Mutation Across Multiple Tumor Types: Correlation Between DNA-based Sequencing and Mutation-specific Immunohistochemistry. Appl Immunohistochem Mol Morphol 2018;26:709-713.

The Comparison of Real-Time PCR and Mutation-Specific Immunohistochemistry in EGFR Mutation Analysis of Non-Small Cell Lung Carcinomas

Year 2024, Volume: 6 Issue: 2, 117 - 125, 30.06.2024
https://doi.org/10.52827/hititmedj.1351295

Abstract

Objective: This study aims to identify activating mutations in the epidermal growth factor receptor (EGFR) gene in patients with non-small cell lung cancer (NSCLC) and to evaluate their correlation with responses to EGFR-tyrosine kinase inhibitors (TKI) treatment. This study aims to identify activating mutations in the epidermal growth factor receptor (EGFR) gene in patients with non-small cell lung cancer (NSCLC) and to evaluate their correlation with responses to EGFR-tyrosine kinase inhibitors (TKI) treatment. We conducted a comparative analysis of Real-Time PCR and immunohistochemistry to detect EGFR mutation status in non-small cell lung cancer patients, focusing on the sensitivity, specificity, and predictive values of immunohistochemistry.
Material and Method: We evaluated 788 non-small cell lung cancer samples which were analyzed for EGFR mutation status by RT-PCR. We detected 126 EGFR mutated cases among these patients. We evaluated mutation-specific EGFR immunohistochemistry directed towards the exon 19 deletions (15 bp E746-A750) and exon 21 point mutation (L858R) to the 47 EGFR mutated patients histologic material and cell blocks of cytologic specimens.
Results: 32 of the 47 cases (68%) had exon 19 deletion, 14 of them (30%) had point mutation in exon 21, and one of them (2%) showed exon 18 mutation. EGFR exon 19 (15 bp E746-A750 deletion) antibody showed a sensitivity of 100%, specificity of 40%, negative predictive value of 100%, and positive predictive value of 78%. The sensitivity of the exon 21 (L858R point mutation) antibody was 93%, specificity was 91%, negative predictive value was 97% and positive predictive value was 82%.
Conclusion: Our investigation indicates that mutation-specific EGFR immunohistochemistry has demonstrated a notable sensitivity and specificity for exon 21. However, while sensitive, the exon 19 (15 bp E746-A750 deletion) antibody lacked specificity. While positive immunohistochemical staining may suggest the presence of an EGFR mutation, making the patient potentially eligible for TKI treatment, it should not be the sole determinant. If immunohistochemistry results are negative, it is essential to resort to molecular tests to ensure accurate diagnosis and appropriate therapeutic guidance. With evolving diagnostic landscapes, it is crucial to harness both IHC and molecular techniques judiciously for optimal patient care.

Project Number

1146

References

  • Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021;71:209-249.
  • Jakobsen E, Olsen KE, Bliddal M, et al. Forecasting lung cancer incidence, mortality, and prevalence to year 2030. BMC Cancer 2021;21:985.
  • George J, Lim JS, Jang SJ, et al. Comprehensive genomic profiles of small cell lung cancer. Nature 2015;524:47-53.
  • Ruiz-Cordero R, Devine WP. Targeted Therapy and Checkpoint Immunotherapy in Lung Cancer. Surg Pathol Clin 2020;13:17-33.
  • Alexander M, Kim SY, Cheng H. Update 2020: Management of Non-Small Cell Lung Cancer. Lung 2020;198:897-907.
  • Liu X, Wang P, Zhang C, Ma Z. Epidermal growth factor receptor (EGFR): A rising star in the era of precision medicine of lung cancer. Oncotarget 2017;8:50209-50220.
  • Yu J, Kane S, Wu J, et al. Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clin Cancer Res 2009;15:3023-3028.
  • Kitamura A, Hosoda W, Sasaki E, et al. Immunohistochemical detection of EGFR mutation using mutation-specific antibodies in lung cancer. Clin Cancer Res 2010;16:3349-3355.
  • Rossi G, Ragazzi M, Tamagnini I, et al. Does Immunohistochemistry Represent a Robust Alternative Technique in Determining Drugable Predictive Gene Alterations in Non-Small Cell Lung Cancer? Curr Drug Targets 2017;18:13-26.
  • Kozu Y, Tsuta K, Kohno T, et al. The usefulness of mutation-specific antibodies in detecting epidermal growth factor receptor mutations and in predicting response to tyrosine kinase inhibitor therapy in lung adenocarcinoma. Lung Cancer 2011;73:45-50.
  • Simonetti S, Molina MA, Queralt C, et al. Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer. J Transl Med 2010;8:135.
  • Seo AN, Park TI, Jin Y, et al. Novel EGFR mutation-specific antibodies for lung adenocarcinoma: highly specific but not sensitive detection of an E746_A750 deletion in exon 19 and an L858R mutation in exon 21 by immunohistochemistry. Lung Cancer 2014;83:316-323.
  • Prenzel N, Fischer OM, Streit S, et al. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr Relat Cancer 2001;8:11-31.
  • Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129-2139.
  • Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497-1500.
  • Cancer Genome Atlas Research N. Comprehensive molecular profiling of lung adenocarcinoma. Nature 2014;511:543-550.
  • Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947-957.
  • Ettinger DS, Aisner DL, Wood DE, et al. NCCN Guidelines Insights: Non-Small Cell Lung Cancer, Version 5.2018. J Natl Compr Canc Netw 2018;16:807-821.
  • Warth A, Penzel R, Brandt R, et al. Optimized algorithm for Sanger sequencing-based EGFR mutation analyses in NSCLC biopsies. Virchows Arch 2012;460:407-414.
  • Brevet M, Arcila M, Ladanyi M. Assessment of EGFR mutation status in lung adenocarcinoma by immunohistochemistry using antibodies specific to the two major forms of mutant EGFR. J Mol Diagn 2010;12:169-176.
  • Houang M, Sioson L, Clarkson A, et al. EGFR mutation specific immunohistochemistry is a useful adjunct which helps to identify false negative mutation testing in lung cancer. Pathology 2014;46:501-508.
  • Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005;97:339-346.
  • Allo G, Bandarchi B, Yanagawa N, et al. Epidermal growth factor receptor mutation-specific immunohistochemical antibodies in lung adenocarcinoma. Histopathology 2014;64:826-839.
  • Leighl NB, Rekhtman N, Biermann WA, et al. Molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors: American Society of Clinical Oncology endorsement of the College of American Pathologists/International Association for the study of lung cancer/association for molecular pathology guideline. J Clin Oncol 2014;32:3673-3679.
  • Cooper WA, Yu B, Yip PY, et al. EGFR mutant-specific immunohistochemistry has high specificity and sensitivity for detecting targeted activating EGFR mutations in lung adenocarcinoma. J Clin Pathol 2013;66:744-748.
  • Remon J, Majem M. EGFR mutation heterogeneity and mixed response to EGFR tyrosine kinase inhibitors of non small cell lung cancer: a clue to overcoming resistance. Transl Lung Cancer Res 2013;2:445-448.
  • Jakobsen JN, Sorensen JB. Intratumor heterogeneity and chemotherapy-induced changes in EGFR status in non-small cell lung cancer. Cancer Chemother Pharmacol 2012;69:289-299.
  • Fassina A, Corradin M, Zardo D, et al. Role and accuracy of rapid on-site evaluation of CT-guided fine needle aspiration cytology of lung nodules. Cytopathology 2011;22:306-312.
  • Lindeman NI, Cagle PT, Aisner DL, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Mol Diagn 2018;20:129-159.
  • Hasanovic A, Ang D, Moreira AL, Zakowski MF. Use of mutation specific antibodies to detect EGFR status in small biopsy and cytology specimens of lung adenocarcinoma. Lung Cancer 2012;77:299-305.
  • Uruga H, Mino-Kenudson M. ALK (D5F3) CDx: an immunohistochemistry assay to identify ALK-positive NSCLC patients. Pharmgenomics Pers Med 2018;11:147-155.
  • Luk PP, Selinger CI, Mahar A, Cooper WA. Biomarkers for ALK and ROS1 in Lung Cancer: Immunohistochemistry and Fluorescent In Situ Hybridization. Arch Pathol Lab Med 2018;142:922-928.
  • Loo E, Khalili P, Beuhler K, et al. BRAF V600E Mutation Across Multiple Tumor Types: Correlation Between DNA-based Sequencing and Mutation-specific Immunohistochemistry. Appl Immunohistochem Mol Morphol 2018;26:709-713.
There are 33 citations in total.

Details

Primary Language English
Subjects Pathology, Gene and Molecular Therapy
Journal Section Research Articles
Authors

Çisel Aydın Meriçöz 0000-0002-4541-793X

Gaye Güler This is me 0000-0001-6112-8719

Sevgen Önder 0000-0002-5523-0669

Project Number 1146
Publication Date June 30, 2024
Submission Date September 8, 2023
Acceptance Date April 29, 2024
Published in Issue Year 2024 Volume: 6 Issue: 2

Cite

AMA Aydın Meriçöz Ç, Güler G, Önder S. The Comparison of Real-Time PCR and Mutation-Specific Immunohistochemistry in EGFR Mutation Analysis of Non-Small Cell Lung Carcinomas. Hitit Medical Journal. June 2024;6(2):117-125. doi:10.52827/hititmedj.1351295