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Attualit e Prospettive in Immuno-Oncologia
Roma, ISS, 4 Novembre 2016
Criteri di valutazione della risposta: monitoraggio
immunologico e nuovi marcatori di efficacia
PAOLA NISTICO M.D.
Laboratorio di ImmunologiaIstituto Nazionale Tumori Regina Elena
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di
Sanit
Da un modello Cell-Oriented ad una visione Tumor-Oriented della trasformazione neoplastica
Risposta immunitaria e microbiota
Risposta immunitaria al tumore e microambiente tumorale
Extracellular matrix (ECM)
ECM
ECM
Modified by Adams et al., Nature Rev 2015 and Lee and Mazmanian, Science 2010
4 Novembre 2016
Istituto Superiore di
Sanit
Chen and Mellman, Immunity 2013
La generazione dellimmunit anti-tumorale un pro cesso ciclico
The Cancer-Immunity Cycle 4 Novembre 2016
Istituto Superiore di
Sanit
Cancer-Immunity Cycle:
Fattori Stimolatori e Inibitori
Chen and Mellman, Immunity 2013
4 Novembre 2016
Istituto Superiore di
Sanit
Mahoney et al., Nature Rev, 2015
Lattivazione delle cellule T un processo che coi nvolge segnali attivatori ed inibitori
4 Novembre 2016
Istituto Superiore di
Sanit
CTLA-4 e PD-1 operano a differenti stadi nella risp osta immunitaria anti-tumorale
Luke, Oncotarget 2015
Linfonodo Periferia/TumoreCellule T Nave
Cellule TumoraliCellule T inattivate
Cellule del sistema immunitario(macrofagi, cellule dendritiche)
CTLA-4
PD-L1
PD-L2
PD-L1
PD-L1
PD1
PD1
PD1
Cellule T inattivate
4 Novembre 2016
Istituto Superiore di
Sanit
I meccanismi inibitori di CTLA -4 e PD-1 sono distinti
Pardoll DM Nat Rev. Imm. 2012
CTLA-4 blocca completamente la co-stimolazione CD28 -mediata
Leffetto inibitorio del PD-1 sul TCR e sul CD28 indiretto, meno completo e pi lento
La nuova era degli immunocheckpoint inibitori
Loncologia incontra limmunologia
Breaking the immune -tolerance 4 Novembre 2016
Istituto Superiore di
Sanit
Blocco degli immunocheckpoint per rinvigorire la risposta
linfocitaria T contro il tumore
Sharma and Allison, Science 2015
+
Meccanismi di azione di PD1, PDL1 e PDL2
Nguyen and Ohashi, Nat Rev Immunol 2015
PD-1 PDL1 PDL2
PD-1 PD-L1 PD-L2
Naive T cells
CTLA-4
+
Activated T cells + +
Exhausted T cells +
NK +
T Reg
MDSC
Endothelial cells
B cells
DC
APC
Fibroblast/CAF
Tumor cells
++
+
++
++
+
+
+
-/+ +
+
-/+
Stromal cells +
+
-/+
Macrophages ++
-/+
+ +
Pattern di espressione
-/+ -/+
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di Sanit
To contribute to the success of Immuno-Oncology
we need to focus on the immune assessment
modalities and sites, both systemic and importantly
intratumoral
Refining immune endpoints to better design
clinical trials represents a high priority challenge
Sharma P, Nat Rev. Cancer, 2011
Clinical trial concepts
Progress to identify new and validate current biomarker candidates has been
limited by the use of unstandardized assays that provide limited data and
variable results.
Monitoring
Mechanisms
Standardized monitoring techniques
Understanding immunogenic mechanisms and designing of efficient combined immunotherapy
Immuno-modulatory drugs Molecules
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di
Sanit
The development of immune monitoringassays is essential to determine the immuneresponses in clinical trials of novelimmunotherapies and targeted drugs
To discover the immune profiles of disease, leading to new
biomarkers of diagnosis, prognosis, and response to therapy
To transfer these therapies to standard of care in oncology
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di
Sanit
1. Standardized immune assays leading to
reproducible results
2. A repository of immune monitoring data
available for mining
Two prerequisites are:
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di
Sanit
Assays must have:
Sufficient sensitivity and specificity to be clinically useful
Good intra- and inter-assay precision
Linearity over a biologically useful range
Robust performance across minor lab variables
This will allow:
Ability to detect small changes that may characterize a disease state
Potential for assays to become clinical diagnostics
Standardized immune monitoring
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di
Sanit
MIATA
A project for minimal Information about T cell assays
Many assays to evaluate the nature, breadth and
quality of antigen-specific T cell responses are
currently applied in human medicine
Experiments conducted by more than 100 labs have
generated the MIATA guidelines
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di
Sanit
Britten et al, Immunity 2012
Minimal Information on the sample: Donors: report all available donor information for interpretation of the data
The Source and Processing: report the source, the conditions of transport and the median time of sample collection
Cryopreservation and storage
Cell Counting: before freezing, after thawing and after overnight resting
Minimal Information on the Assay: Medium/serum
The assay: report details about assay procedure including all reagents used
Controls
Data acquisition: Equipment and Software
Acquisition Strategy and Gating
The (interpretation of) results: Raw data: provide the means, medians and ranges for both background and antigen-
specific reactivity levels
Response determination, statistical tests and empirical rules
The laboratory environment: General Laboratory operation: GMP, GLP, exploratory research etc.
Laboratory procedure standardization
Status of Assay Qualification and validation
Corso ECMAttualit e prospettive in
Immuno-oncologia
Evaluation of the T-cell response will require the use of spe cific assays toaddress issues related to T-cell frequency, trafficking an d function, aswell as issues related to potential immune-escape mechanis ms that areenlisted by the tumour to evade the T-cell response
Yee et al. Nature Rev Cancer 2002
Monitoring the anti-tumour T-cell responseCorso ECM
Attualit e prospettive in Immuno-oncologia
Flow Cytometry for phenotypic analysis, including leukocyte subset analysis and Intracellular Cytokine staining (ICS)
Peptide/MHC Multimer staining
Enzyme-linked Immunospot Assay (ELISPOT)
Bio-plex Multiplex Immunoassay
TCR V Repertoire Analysis using Flow cytometrystaining, spectratyping and sequencing
Validated assays in immune monitoring
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di
Sanit
Multiparameter Flow cytometry assay for phenotypic analysis and intracellular cytokine staining (ICS)
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di Sanit
La catena pesante della molecola HLAmodificata per contenere un sito dibiotinilazione e la b2-microglobulinavengono espresse in un sistemaprocariotico.
In seguito a purificazione delle proteinericombinanti viene aggiunto il peptide.
Una reazione enzimatica inducebiotinilazione della molecola e laggiuntadi streptavidina, che contiene quattro sitidi legame per la biotina, induce laformazione del complesso tetramerico.
La marcatura della streptavidina con uncomplesso fluorescente permettelidentificazione tramite analisi percitometria a flusso dei linfociti T dotatidi TCR specifico per il complesso HLA-peptide
HLA class I MultimersCorso ECM
Attualit e prospettive in Immuno-oncologia
Yee et al. Nature Rev Cancer 2002
ex-vivo
Melan-Ain vitro
expansion
PRE T42 T84 T105
1.15%1.04%0.05%0.04%
7.5% 17.0% 44.0% 70.0%
CD8
A2/
Mel
an-A
te
tram
er
0.45% 0.22%
70.0% 90.0%
POST(3 months)
POST(17 months)
Monitoring of a HLA-A2 melanoma patient by A2/Melan-A Multimer staining
HLA-A2/MULTIMER
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di Sanit
ELISPOT(enzyme-linked immunospot assay) CVIA (computer video image assisted)
Short-term sensitizedIFN- Elispot assay
Pre Post
T2 +Melan-A
T2 empty
Corso ECMAttualit e prospettive in
Immuno-oncologia
Bio-Plex Multiplex Immunoassay
Cytokines, chemokines and grow factors detection by a bead-based flow cytometric platform
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di Sanit
MASS CYTOMETRYBased on the use of monoclonal antibodies labelled with stable
isotopes
ADVANTAGES
Up to 100 independent detection channels
Reproducibility between sample
Absolute quantification of target molecules
Overcomes the compensation step
USES
Analysis of phenotype
Analysis of signaling pathways
Effects of inhibitors on feedback signaling and intercellular comunication
Corso ECMAttualit e prospettive in
Immuno-oncologia
4 Novembre 2016
Istituto Superiore di Sanit
To contribute to the success of immuno-
oncology we need to focus on the immune
assessment modalities and sites, both
systemic and importantly intratumoral
The immune contexture
During the past two decades, it has become clear th at the interaction between tumours and
their microenvironment (including stromal, endothel ial and immune cells) is crucial not only
during oncogenesis and tumour progression but also in the context of anticancer therapies
Fridman WH, Nature Rev, Cancer, 2012
TLS conduttori della risposta immunitaria adattativa e sistemica contro il tumore
I tumori sono infiltrati dalle cellule del sistema immunitario
Le strutture linfoidi terziarie (TLS) e linfiltrato linfocitario: fattori prognostici
favorevoli nel Non-Small Cell Lung Cancer
Modified by Dieu-Nosjean et al., Immunological Rev, 2016
4 Novembre 2016
Istituto Superiore di
Sanit
Corso ECM
Attualit e prospettive in Immuno-oncologiaTechnologies currently used to assess the potential
immune biomarkers
TUMOR
Type of Assay Tumor-derived Biomarker Immune cell-derived Biomarker
Protein (IHC)
Genomics
Transcriptomics
Function
PD-L1, PD-L2, TAAs
Next-generation sequencing
or Whole exome sequencing
for mutation load,
neoantigens, MMR genes
CD8, CD4, CD3, CTLA-4, PD-1,
LAG-3, CD45RO, CD25, FOXP3,
CD127, CD11B, CD57, CD68,
co-stimulator(s)
T and/or B cell receptor deep
sequencing
Multigene signatures Multigene signatures,
epigenomics
Proliferative marker: Ki67 Ki67, granzyme B, IFN-
by Ma W., J. of Hematology &&&& Oncology 2016
Corso ECM
Attualit e prospettive in Immuno-oncologia
4 Novembre 2016Istituto Superiore di Sanit
Cell free
genomics or
proteomics
Cell based
ctDNA, ctRNA, miRNA,
exosomes
Circulating tumor cells
miRNA, serological biomarkers
(soluble proteins, autoantibodies,
cytokines or chemokines);
HLA haplotyping
Absolute lymphocyte counts
(ALC), Eosiniphils,
Neutrophil/Leukocyte ratio,
MDSC, Th17,
Tumor-specific Immune cells
BLOOD
by Ma W., J. of Hematology &&&& Oncology 2016
Technologies currently used to assess the potential immune biomarkers
Type of Assay Tumor-derived Biomarker Immune cell-derived Biomarker
Corso ECM
Attualit e prospettive in Immuno-oncologia
4 Novembre 2016Istituto Superiore di Sanit
Flow and
mass cytometry
Antigen-
specific T cell
monitoring
Phenotypic and functional characterization of different immune
cells:
Activated T cells: CD3, CD4, CD8, Ki67, CTLA-4, PD-1, LAG-3,
TIM-3, ICOS
Regulatory T cells: CD3, CD4, CD25, FOXP3, CD127, Ki67,
CD45RA
MDSC: CD14+HLA-DRlow
Detection and characterization of circulating tumor cells (CTC)
Characterization of tumor-antigen specific T cells:
Sorting, Cloning, Binding avidity, Antigen-specific TCR
Functional assessment of response by tumor antigen-
specific T cells:
HLA-class I and II tetramer, ELISPOT, Polyfunctional ICS,
Natural tumor recognition
BLOOD
by Ma W., J. of Hematology &&&& Oncology 2016
Technologies currently used to assess the potential immune biomarkers
TUMOR
Corso ECM
Attualit e prospettive in Immuno-oncologia
4 Novembre 2016Istituto Superiore di Sanit
Topalian, Nature Rev Cancer 2016
Multifactorial biomarkers of clinical response to PD-1 pathway blockade
Corso ECM
Attualit e prospettive in Immuno-oncologia
4 Novembre 2016Istituto Superiore di Sanit
Topalian Nature Rev 2016
Pitfalls of using PD -L1 immunohistochemistry as a biomarker test for anti-PD -1/PD-L1 therapy
Corso ECM
Attualit e prospettive in Immuno-oncologia
Mutational load as a potential biomarker for response
A cancer mutation can produce a T cell neoantigenic peptide t hat can confer de
novo peptide binding to MHC molecules.
The antigenicity of a neopeptide is not related to its functi on and a passenger
mutation can be a good tumor antigen.
Tumors with a greater mutational load could possess more neo antigens and thus
the patients may possess a larger repertoire.
Lawrence, Nature 2013
The odds of immunotherapy success
Schreiber R, Science 2015
4 Novembre 2016
Istituto Superiore di
Sanit
4 Novembre 2016Istituto Superiore di Sanit
Modified by Chen, Cancer Discov 2016
Immune profiling in early on-treatment biopsies is predictive of response to sequential CTLA-4 and PD-1 blockade
Corso ECM
Attualit e prospettive in Immuno-oncologia
4 Novembre 2016Istituto Superiore di Sanit
Optimal time points for tumor- and blood -
based biomarker measurement
To capture the kinetics of the immunologic
phenomena, extensive serial samplings of blood at
every cycle with more frequent collections during the
first cycle may be warranted
We are (fortunately) not alone
Y K Lee, and S K Mazmanian Science 2010
In humans, trillions of bacteria are distributed in complex and site-specific
communities on the skin and at mucosal surfaces, and the larg est community
is found in the distal gut
Round, Nat Rev Immunol 2009
Immunological dysregulation associated with dysbios is of the microbiota
A healthy microbiota contains a balanced composition of man y classes of bacteria
In condition of dysbiosis reduction of symbionts, increase of pathobionts and inflammation
The efficacy of cancer immunotherapy relies on gut
microbiota
Vetizou M, Science Nov 2015
4 Novembre 2016
Istituto Superiore di
Sanit
Certain gut microbiota determine the level of effic acy and toxicity during CTLA -4 checkpoint blockade.
Jonathan M. Pitt et al. Cancer Res 2016;76:4602-460 7
4 Novembre 2016
Istituto Superiore di
Sanit
Our experience
Peptide (Melan-A/gp100)-based vaccine in combination with chemotherapy (DTIC) in 10
melanoma patients (phase I pilot clinical trial)
Peptide (Melan-A/NY-ESO-1)-based vaccine in combination with chemotherapy (DTIC) in 35
melanoma patients (phase II randomized clinical trial)
Corso ECMAttualit e prospettive in
Immuno-oncologia
MELANOMA PATIENTS(admitted in the Melanoma Unit)
Sera and Peripheral Blood CollectionNeoplastic Lesions paraffin embedded snap frozen
SerumPeripheral Blood
Lymphocytes immunohistochemical analysis
(tumor phenotype; HLA class I expression; T cell infiltrate
localization and phenotyping)Ex-vivo analysis of
T cell response
Antibody Response
Analysis of in-vitro cultures
T cell cloning and analysis of TCR repertoire
IDENTIFICATION OF PREDICTORS OF
RESPONSE
Tumor immune microenvironment
Corso ECMAttualit e prospettive in
Immuno-oncologia
IRE Immune Monitoring AssaysFlow cytometry:
Multiparametric immunophenotyping of T (CD4, CD8, ) and B cells
Enumeration of ex-vivo Ag-specific CD8+ T cells by Peptide/MHC Tetramers staining in PBMC, TIL and TILN
Analysis of differentiation phenotype for T and B cells
Analysis of immune checkpoint inhibitory receptors (PD-1, LAG-3, TIM-3) and PD1 ligand (PD-L1) expression
Intracellular staining (ICS) for evaluation of cell ular polyfunctionality
ELISPOT assay CVIA
Ag-specific T-cell lines and clones in vitro expanded
TCR V Repertoire analysis by sequencing Immune cell infiltration
Tumor immune microenvironment
Ag-specific-line -clone
ELISA for antibody response
Ex-vivo T cells
51Cr release cytotoxicity assay
Bio-plex Multiplex Immunoassay for multiple cytokin es detection
BioBanking of tumor tissue, PBMC, serum/plasma, TIL , TILN
Tertiary lymphoid structures CD3 CD20
Exploring the influence of DTIC on the functional activity of CD8+ T cells
Generation of a multitude of Melan-A+ T-cell clones
Characterization of phenotype and TCR repertoire of
T-cell clones
Analysis of the lytic activity
Analysis of co-stimulatory and inhibitory receptor s
4 Novembre 2016
Istituto Superiore di
Sanit
Polyfunctionality
Activation of AKT in absence of CD28/C27 co-stimula tion
Immune checkpoint
Searching for biomarkers in patients who benefit from
chemoimmunotherapy treatment
Franzese, Palermo et al. Polyfunctional Melan-A-specific tumor-reactive CD8+ T cells elicited by Dacarbazine treatment before peptide-vaccination depends on AKT activation sustained by ICOS. OncoImmunology 2016
Responder patients
TCR diversity enlargement
Arm2
Proliferative capability
Palermo et al, Cancer Res, 2010
Up-regulation of ICOS co-stimulatory molecule
Limmunoterapia dei tumori personalizzata
Gubin J Clin Invest 2015
Cellule linfocitarie T specifiche per neo-antigeni mutati
Vaccini terapeuticiTrasferimento adottivo di
cellule linfocitarie T
Corso ECM
Attualit e prospettive in Immuno-oncologia
4 Novembre 2016Istituto Superiore di Sanit