HistoCyte Laboratories Ltd Progesterone Receptor: The neglected breast receptor! Dr Ian Milton & Colin Tristram November 2018 UKNEQAS Autumn meeting
Introduction Progesterone is an important prognostic and predictive marker for endocrine therapy. The poor relation, not always tested not always thought about. In this presentation we will afford it some attention: The biology of progesterone receptor(s) Review the antibody clones The use of controls The development of Progesterone Receptor Analyte Control DR The focus of HistoCyte Laboratories.
PR in endocrine biology In breast development ER is responsible for the growth of the ducts whereas PR is responsible for the growth of lobules. PR has a mitogenic effect in breast, driving proliferation. PR exists in several different isoforms. Work with knock out mice demonstrated that PR-B rather than PR-A is primarily needed for ductal extension and alveologenesis 1. PR-C is appears to have no classical transcriptional activity, but when present it can enhance PR activity in breast cancer cells 2 In the uterus PR-C functions as a dominant inhibitor of PR-B 3 Image adapted from: Karamouzis MV et al. Targeting Androgen/Estrogen Receptors Crosstalk in Cancer. Trends Cancer. 2016 Jan;2(1):35-48. 1. Conneely OM, et al. Reproductive functions of the progesterone receptor isoforms: lessons from knock-out mice. Mol Cell Endocrinol. 2001; 1(1 2): 97 103. 2. Wei LL, et al. An N-terminally truncated third progesterone receptor protein, PR(C), forms heterodimers with PR(B) but interferes in PR(B)-DNA binding. J Steroid Biochem Mol Biol. 1997; 62(4):287 297 3. Condon JC, et al. Up-regulation of the progesterone receptor (PR)-C isoform in laboring myometrium by activation of nuclear factor-kappab may contribute to the onset of labor through inhibition of PR function. Mol Endocrinol. 2006; 20(4):764 775
PR Structure PGR-B PGR-A/B PR gene 1 2 3 4 5 6 7 8 PR-B PR-A PR-C N-Terminal transactivating domain DBD Hinge Ligand Binding domain 250KDa 150KDa 114KDa 75KDa 114KDa 94KDa 50KDa PR assessed to determine presence of intact estrogen receptor pathway. Not as good an indicator as ER Does recognising different PR isoforms matter?
What does it mean in IHC? Phenotype Incidence % (n) Response % (n) ER+/PR+ 58 (29,000) 77 (22,230) ER+/PR- 23 (11,500) 27 (3,105) ER-/PR+ 4 (2,000) 46 (920) ER-/PR- 15 (7,500) 11 (825) Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical anlaysis. Mod Pathol 1998; 11(2):155 ER+ with PR+ very strong predictor of response! ER only ~60% response rate. The importance of testing for PR is underlined by the ER-/PR+. ~50% of ER-/PR+ respond to Tamoxifen. Number similar to the ER-/PR- are these missed ER/PR cases? HER2 status unknown
PR antibodies and Vendor Share of PR testing by vendor/clone 1E2 1.4% 0.5% PgR 636 PgR 1294 18.6% 1A6 44.8% 1.4% 2.3% 16 16+SAN27 1E2 26.2% SP2 Y85 5.0% RUN 119 Oct 2017 3 antibody clones dominate 90% of the market
PR antibodies and NEQAS results Vendor Clone Passed Failed % Pass Agilent PgR 636 29 12 71% PgR 1294 2 1 67% 1A6 3 2 60% Leica 16 54 4 93% 16+SAN27 11 0 100% Ventana 1E2 78 21 79% SP2 2 1 67% Cell Marque Y85 0 1 0% RUN 119 Oct 2017 Clone 16 RTU appears to perform best PR EQA pass rate are generally lower than ER ER Run: 118 PR Run: 119 No pass % No Pass % 79 94 28 68 38 97 33 94 21 100 34 82 29 86 65 77 19 84 Average% 94.0 80.4 No standardised control material. Does recognising PR-A and B matter? SAN27 PR-B only Combination with 16 works well (n=11)
The importance of controls Most if not all instruments treat slides independently. Batch controls are not sufficient the control or any of the slides can be exposed to a variable. False negatives the greatest risk. Tissues are great but often affected by protein heterogeneity and artefacts associated with fixation and processing. No two tissues are the same content different from one to another. Fixation and process can differ between them (Bx vs Rx). Nitta et al. Diagnostic Pathology 2012, 7:60
Use of Quality Controls Internal positive control Torlakovic et al Appl Immunohistochem Mol Morphol 2015;23:1-18
The importance of external controls and EQA Control Low Medium High Capture All L M Comment X X X Yes Best overall effectiveness X X X Yes No No Mid expression maybe high High expression maybe overstained Low expression missed, High overstained Low expression missed Mid expression H possibly missed High expression maybe under stained Cell line Controls and EQA Cells are admissible to NEQAS, However, to get high score at least one positive tissue is required. Very hard to find tissue that has reproducible expression levels Characterised external material and regular EQA help reduce the risk from working in isolation Cell lines offer a method of producing highly reproducible levels of biomarker expression Cell lines are ideal for on-slide routine controls
Progesterone Receptor Analyte Control DR Product Development Objectives Identify positive and negative cells as well as low/mid expressing cell lines to provide sensitivity. Verification - Reproducibility of biomarker expression in each cell line both slide-slide and batch-batch Validation -Cell lines can visually indicate the failure of the assay, by conducting a series of forced-fails Stability - Determine estimated shelf-life of the product Negative Mid/Low Mid/High High
Progesterone Receptor Analyte Control DR Verification Good consistency in biomarker expression across all batches for each cell line. Most visible variation seen in the low expression cell line. Creates tolerance of performance (The graph shows the ranges of H-Scores recorded for each cell line and the images correspond to the graph (x200): top-bottom: high, moderate, low and negative biomarker expression)
Progesterone Receptor Analyte Control DR Parameter Function Staining Result 2μm Section Thinner than standard Weaker 6μm Section Thicker than standard Slightly stronger No Deparaffinisation No wax removal Little change No Retrieval No cell conditioning Much weaker 8 minutes CC1 Shorter conditioning than standard Considerably weaker 92 minutes CC1 Longer conditioning than standard Little change 64 minutes CC2 Different conditioning reagent Weaker 4 minutes Ab incubation Shorter incubation than standard Considerably weaker 30 minutes Ab incubation Longer incubation than standard Slightly stronger 60 minutes Ab incubation Longer incubation than standard Slightly stronger Negative: Under retrieved/no retrieval. Low/Mid: No Dewax
H-Score Progesterone Receptor Analyte Control DR 500 450 400 350 300 250 Stability: Blocks Little fluctuation seen in the average H- Scores of the cores in the aged-block. It is within performance tolerance? 200 150 100 50 0 0 10 20 30 40 50 60 70 Day Negative Low Moderate High Negative Control Low Control Moderate Control High Control
H-Score Progesterone Receptor Analyte Control DR 600 500 400 Stability: pre-cut slides Little fluctuation seen in the average H- Scores. 300 200 100 0 0 10 20 30 40 50 60 70 Day Negative Low Moderate High Negative Control Low Control Moderate Control High Control
Field Trials HCL: 1E2 on Roche/Ventana Ultra Site A: PGR636 on the Leica B3 Site B: 1E2 on Roche/Ventana Ultra 10 sites performed premarket assessments. EQA bodies as well as hospital laboratories. All were in agreement except 1 Aesthetics and interpretations were the most important factors. Site C: 16 on the Leica Bond
ER Analyte Control DR Negative Low Mid High ER Roche (SP1) Ventana
PathVysion Vysis 4B5 Ventana Ultra HER2 Analyte Contorl DR 0/Non-amplified 1+/Non-amplified 2+/Equivocal 3+/Amplified
PD-L1 Analyte Contorl DR SP142 (Ventana/Roche) SP263 (Ventana/Roche) 22C3 PharmDx (Dako) E1L3N (Cell Signalling Technologies Clone on Ventana) PD-L1 Analyte Control DR
In development: ROS1 Analyte Control DR Negative (very) Low: FIG-ROS1 High SLC34A2-ROS1 ROS1 Fusions Associated with Lung Adenocarcinoma
In development ALK Analyte Control DR : NPM-ALK and EML4-ALK Negative Negative Weak/Positive Positive Lymphoma NPM-ALK Assay Ventana CONFIRM anti- ALK1 (ALK01) Primary Antibody Lung EML4-ALK Assay Ventana ALK (D5F3) CDx Assay 1 2 3 4 Cell lines very easily able to determine whether the assay is suitable for lymphoma or Lung. Negative Positive (intermediate) Positive (strong) Positive (very strong) Negative WT ALK EML4-ALK NPM- ALK
Mismatch Repair Controls MLH1 test A B C D MSH2 test A B C D Cell line A: Loss of MLH1/PMS2 due to MLH1 mutation or promoter hypermethylation Cell line B: Loss of MSH2/MSH6 due to MSH2 mutation PMS2 test MSH6 test Cell line C: Loss of MSH6 expression due to MSH6 mutation A B C D A B C D Cell line D: Loss of PMS2 expression due to PMS2 mutation
Products Product Name HPV/p16 Analyte Control DR (Four cores with a dynamic range of HPV gene copies) HPV/p16 Analyte Control (Three cores with a standard range of HPV gene copies) ALK-Lung Analyte Control (Two cores one positive and one negative for the EML4-ALK translocation) ALK-Lymphoma Analyte Control (Two cores one positive and one negative for the NPM-ALK translocation) Breast Analyte Control (Two cores, one positive for HER2, ER and PR. The other negative) Breast Analyte Control DR (Five cores with a dynamic range of expression of HER2, ER and PR) Format Slide(2) Slide(5) Block Slide(2) Slide(5) Block Slide(2) Slide(5) Block Slide(2) Slide(5) Block Slide(2) Slide(5) Block Slide(2) Slide(5) Block Code HCL001 HCL002 HCL003 HCL004 HCL005 HCL006 HCL007 HCL008 HCL009 HCL010 HCL011 HCL012 HCL013 HCL014 HCL015 HCL016 HCL017 HCL018 Product Name PD-L1 Analyte Control DR (4 cores with a dynamic range of PD-L1 expression) ROS1 Analyte Control (Two cores one positive and one negative for ROS1 translocation) HER2 Analyte Control DR (Four cores, 0, 1+ (both non-amplified), 2+ (equivocal) and 3+ (amplified)) Estrogen Receptor Analyte Control DR (Four cores: negative, low, intermediate and high) Progesterone Receptor Analyte Control DR (Four cores: negative, low/intermediate, intermediate/high and high) Format Slide(2) Slide(5) Block Slide(2) Slide(5) Block Slide(2) Slide(5) Block Slide(2) Slide(5) Block Slide(2) Slide(5) Block Code HCL019 HCL020 HCL021 HCL022 HCL023 HCL024 HCL026 HCL027 HCL028 HCL029 HCL030 HCL031 HCL032 HCL033 HCL034
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