Integrazione in Anatomia Patologica dei markers predittivi nel NSCLC: l esperienza di PD-L1

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2 Integrazione in Anatomia Patologica dei markers predittivi nel NSCLC: l esperienza di PD-L1 Dipartimento di Sanità Pubblica Università degli Studi di Napoli Federico II Dott. Antonino Iaccarino

3 PDL1-Mechanism Restifo NP et al : Nat Rev Cancer Feb;16(2):121-6.

4 Adaptive vs Innate Restifo NP et al : Nat Rev Cancer Feb;16(2):121-6.

5 Lung Cancer Subtypes Lung Cancer Non-Small Cell (65-85%) Small Cell (15-35%) No Driver Mutation (80%) PD-L1 Oncogenic Driver Mutation (20%) Nonsquamous (60-80%) Squamous (20-40%) ALK (2-5%) EGFr (15-18%) Adenocarcinoma (50-70%) Large Cell (10-30%)

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7 PDL1-CD8-Mutational load

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9 A Biomarker Is Defined As: European Medicines Agency: Biomarkers are tests that can be used to follow body processes and diseases in humans and animals. They can be used to predict how a patient will respond to a medicine or whether they have, or are likely to develop, a certain disease 2 1. FDA Guidance for Industry. Accessed March 2, European Medicines Agency. Accessed March 2, 2015.

10 A Biomarker Is Defined As: Food and Drug Administration: A biomarker that is measured in an analytical test system with wellestablished performance characteristics and for which there is an established scientific framework or body of evidence that elucidates the physiologic, toxicologic, pharmacologic, or clinical significance of the test results 1 1. FDA Guidance for Industry. Accessed March 2, European Medicines Agency. Accessed March 2, 2015.

11 companion diagnostics Approval Order Statement Approval for the pd-l1 ihc 22c3 pharmdx. This device is indicated for the following: pd-l1 ihc 22c3 pharmdx is a qualitative immunohistochemical assay using monoclonal mouse anti-pd-l1, clone 22c3 antibody intended for use in the detection of pd-l1protein in formalin fixed, paraffin embedded (ffpe) non-small cell lung cancer (nsclc) tissue using envision flex visualization system on autostainer link 48. Pd-l1 protein expression is determined by using tumor proportion score (tps), which is the percentage of viable tumor cells showing partial or complete membrane staining. The specimen should be considered pd-l1 positive if tps >= 50% of the viable tumor cells exhibit membrane staining at any intensity. Pdl1 ihc 22c3 pharmdx is indicated as an aid in identifying nsclc patients for treatment with keytruda (pembrolizumab)

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15 IL PROTAGONISTA Anticorpo

16 ANTIGENE = antibody generator Epitopo = determinante antigenico IL CO-PROTAGONISTA Antigene

17 TARGET

18 IHC: TECNICA ANCILLARE Evoluzione Definizione della diagnosi cito-istologica Affinamento della diagnosi cito-istologica Indicazioni prognostiche Markers Diagnostici Markers Prognostici Supporto nel trattamento clinico-farmacologico del paziente Timing: Ab mutazione-specifica PD-L1 Markers Predittivi

19 Immunocitochimica = immunoistochimica?

20 Amplificazione del segnale

21 Sistema di rivelazione Sistema Polimerico

22 Antigen Retrieval Chimico ed enzimatico (Ab-dipendente!) Sistemi di smascheramento Significato biochimico

23 Antigen Retrieval Fissazione Citrato/EDTA Enzimatico Necessario?

24 IHC e Marker Predittivi Conoscenza dei meccanismi molecolari Interazione Tecnico/Patologo Approfondimento in letteratura ma attenzione.

25 Specific Considerations Pitfalls Any perceptible tumor cell membrane staining (full or partial) should be considered as positive 40 x

26 Cytoplasm stain, depending on the intensity can completely or partially obscure membrane stain Often the cell membrane can still be scored if The membrane stain is perceived distinct from the cytoplasm stain A clear zone between the nucleus and the cell membrane is present 40 x

27 Guidelines to distinguish tumor associated immune cells and PD-L1 positive immune cells from tumor cells: Histiocytes may have scattered distribution Immune cells may have smaller nuclei than tumor cells 40 x

28 General Considerations Tumor Associated Immune Cells Pulmonary macrophages are present in the alveolar space Macrophages may contain pigmented particles in their cytoplasm Macrophages may stain stronger than the tumor cells 40 x

29 To assess reagents

30 To assess reagents

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32 To monitor differences in processing and embedding include positive and negative in-house control tissue of the same tumor indication in each staining run, in addition to the PD-L1 IHC 22C3 pharmdx Control Cell Line Slide.

33 The ideal positive control tissue provides a complete dynamic representation of weak to moderate cell membrane staining.

34 The ideal negative control tissue gives no staining on tumor cells but contains tumor-associated macrophages/immune cells which may express PD-L1 and offer an internal positive control

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42 ///// A B C D E F G I L M N Lab 1 Lab 2 Lab 3 Lab 4 ab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10 nv Lab 11 Lab 12 Lab casi colorati da 13 laboratori e valutati utilizzando come gold standard il PD-L1 IHC 22C3 Pharma Dx

43 ///// A B C D G L Reference score 0% 0% O,5 0% 0% 0% 0% Messina Caserta L Aquila Ravenna TPS < 1% M ///// E F I N Reference score 10% 80% 30% 5% Messina Caserta L Aquila Ravenna TPS > 1% Torre Annunziata (NA) Campobasso Cagliari Novara Bari San Paolo Roma Forlanini Sicilia Palermo Bergamo Discordanze (11%) Torre Annunziata (NA) Campobasso Cagliari Novara Bari San Paolo Roma Forlanini Sicilia Palermo Bergamo Discordanze (38%)

44 78,2% 81,7% 79,0% Piattaforme A B C D E F G I L M N Concordancy rate 8/11 9/11 6/11 9/11 11/11 9/11 9/11 8/11 10/11 nv 8/10 9/11 10/11 9/11 7/11

45 ISTIOCITI?

46 Doppia IHC Staining valutabile?

47 P63/PD-L1

48 P63/PD-L1

49 TTF1/PD-L1

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51 TTF1/PD-L1

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