Tissue-based Immunohistochemical Biomarker Expression in Malignant Glandular Lesions of the Uterine Cervix: a Systematic Review

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1 Tissue-based Immunohistochemical Biomarker Expression in Malignant Glandular Lesions of the Uterine Cervix: a Systematic Review Sandra Lee MD, FRCPC 1 *, Vikrant V. Sahasrabuddhe, MBBS, DrPH 2 *, Diana Mendoza-Cervantes, BS 2, M. Sarah Rose, PhD 3, Rachel Zhao, B Eng, MLIS 4, and Máire A. Duggan MD, FRCPC 1 *co-first authors Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, Alberta, Canada 2 Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland, USA 3 Research Facilitation and 4 Knowledge Management Department, Alberta Health Services, Calgary, Alberta, Canada

2 Disclosures No financial relationships or conflict of interest to disclose

3 Malignant glandular histotypes of the cervix: 19 primary histotypes per WHO 2014 HPV DNA positive Adenocarcinoma in situ (AIS) Mucinous adenocarcinoma Endometrioid adenocarcinoma Adenosquamous carcinoma HPV DNA negative Atypical lobular endocervical glandular hyperplasia (LEGH) Minimal deviation and gastric type adenocarcinomas (MDA/GAS) Mesonephric carcinoma Serous and clear cell carcinomas

4 Immunohistochemical testing Numerous diagnostic biomarkers available: accuracy uncertain p16 overexpression as a surrogate marker of HPV DNA is a sensitive and specific biomarker in distinguishing HPV positive malignant glandular lesions from normal glandular epithelium and benign lesions of the cervix* Whether IHC expression can discriminate between the different glandular histotypes has not been systematically analysed *Lee S et al. Tissue based immunohistochemical biomarker accuracy in the diagnosis of malignant glandular lesions of the uterine cervix : a systematic review of the literature and meta-analysis. Int J Gynecol Pathol 2016; Oct 31 (epub).

5 Study goal Systematically review literature reporting immunohistochemicallydetected tissue-based biomarker expression amongst glandular histotypes in order to identify differences in biomarker positivity that could have diagnostic utility

6 PICOT framework Population: AIS, MDA/GAS and *invasive adenocarcinomas Index test: biomarker immunohistochemical expression in tissue samples Comparator: atypical LEGH, MDA/GAS and *invasive adenocarcinomas Outcomes: prevalence of positive biomarker expression Timeframe: January 1, 1975 to June 30, 2015 *classified per WHO 2003

7 Methods 2 reviewers screened abstracts of potential reports for study eligibility Eligible articles reviewed and data extracted on: IHC biomarker name, scoring details and positive-negative cut offs Case and comparator histotypes, and sample sizes Number of positive and negative test results Final selection of articles: QUADAS-2

8 Cases and Comparators Cases and comparators were grouped as: 1) Mucinous adenocarcinoma 2) Endometrioid adenocarcinoma 3) Adenosquamous carcinoma 4) Serous-clear cell carcinoma 5) MDA/GAS 6) Mesonephric carcinoma AIS cases were compared to 6 adenocarcinoma groups and to atypical LEGH (7 AIS case-comparators) 6 groups of adenocarcinoma cases were each compared to 5 groups of adenocarcinoma comparators (30 Adenocarcinoma case-comparators)

9 Biomarker positivity/prevalence analysis Combined estimates of individual biomarker positivity were calculated as weighted averages of estimates by histotype reported among the included studies Data was analyzed using unsupervised hierarchical clustering Cluster 3.0 open access software Clustering visualized via heatmaps and dendrograms Java TreeView open access software 50% or more difference in positivity interpreted as diagnostically useful

10

11 56 biomarkers Alpha-SMA: Alpha smooth muscle actin Beclin-1 CA125 CA-IX: Carbonic anhydrase Calretinin CD10 CD44s CD44v3 CD56 CDX2 CEA: Carcinoembryonic antigen Chromogranin Claudin 18 CK20 CK7 D2-40 E Cadherin EMA: Epithelial membrane antigen Epithelial specific antigen ER: Estrogen receptor GATA3 hent1 HIK1083 HNF1beta: Hepatocyte nuclear factor 1 beta hpankomab: humanized PankoMab Keratan sulfate Ki67 L1 Capsid LC3B MCM7: Minichromosome maintainance complex component 7 MMP-2: Matrix metalloproteinase 2 MUC2: Mucin 2 MUC6: Mucin 6 MUC5AC p16 p16+/ki67+ dual stain P40 p53 p63 PAX2: Paired box gene 2 PAX8: Paired box gene 8 pcea: Polypclonal carcinoembryonic antigen PR: Progesterone receptor prb: Retinoblastoma protein ProExC PTEN: Phosphatase and tensin homolog SMA SOD2: Superoxide dismutase 2 SP17: Sperm protein 17 Synaptophysin Telomerase TTF1 Ubiquitin VIL1: Villin 1 Vimentin

12 Case-comparisons AIS # Biomarkers: ranged from 1-20 Most frequently compared: p16 HIK1083 CD10 Adenocarcinoma # Biomarkers: ranged from 1-2 (mesonephric) to 1-36 (all others) Most frequently compared*: p16 HIK1083 CD10 ER PR *except mesonephric case-comparators

13 Adenocarcinoma In situ versus Mucinous adenocarcinoma Biomarker (case vs. comp positivity) Alpha SMA (0.00 vs. 1.00)

14 Mucinous adenocarcinoma versus Serous-Clear Cell carcinoma Biomarker (case vs. comp positivity) CEA (0.73 vs. 0.00) p53 (0.37 vs. 0.89)

15 Adenosquamous carcinoma versus Minimal deviation/gastric type adenocarcinoma Biomarker (case vs. comp positivity) Chromogranin (0.00 vs. 0.60) HIK1083 (0.00 vs. 0.76) p16 (0.94 vs. 0.28)

16 AIS case-comparators: 8 biomarkers with a positivity difference of 50% or more Cases versus comparators Atypical LEGH Mucinous Endometrioid Adenosquamous Serousclear cell MDA/GAS Mesonephric AIS HIK1083 Alpha- SMA* PAX8 VIL1 CEA p53 Alpha-SMA* HIK1083 p16 p53 CD10 : none with >50% difference in positivity *difference=100%

17 Adenocarcinoma case-comparators: 12 biomarkers with a positivity difference of 50% or more Mucinous Endometrioid Casecomparator Adenosquamous Serous- Clear cell MDA/GAS Mesonephric Mucinous p63 CEA, p53 Endometrioid Adenosquamous PR PR CEA, PR Claudin18, HIK1083, p16 Chromogranin, HIK1083, MUC6, p16, PR, Vimentin Chromogranin, HIK1083, p16 Calretinin CD10 CD10 Serous- Clear cell CEA p53 CEA PR CEA, HIK1083, PR (no data) MDA/GAS Claudin18, HIK1083, p16 Chromogranin, HIK1083, MUC6, P16, PR, Vimentin Chromogranin, HIK1083, p16 CEA, HIK1083, p16 Calretinin, CD10 Mesonephric Calretinin, CD10 CD10 Calretinin, CD10 : none with >50% difference in positivity

18 Conclusions and next steps Biomarker expression is understudied 15 diagnostically useful biomarkers 6/7 AIS case comparators 21/30 Adenocarcinoma case comparators Validate utility of the 15 biomarkers Standardized and consistent processing, scoring, cut offs, and more Generate algorithmic-based biomarker panels for each case-comparison Investigate knowledge gaps, identify and validate additional/new biomarkers

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