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Emerging Tissue and Serum Markers for Immune Checkpoint Inhibitors

Kyong Hwa Park MD, PhD

Medical Oncology

Korea University College of Medicine

Contents

• Immune checkpoint inhibitors in clinical practice

• Biomarkers for immune checkpoint inhibitors

- Tissue or blood based multi-level assays

• Immunotherapy in breast cancer: recent signatures

• Emerging biomarkers

Atezolizumab(Genentech/Roche)

Current Approval Status of ITA by USFDA

Immunologic Armaments in Oncology Clinic

Anti-CTLA4Anti-CD137 agonistAnti-OX40 agonistAnti-CD27 agonist

IL-2IL-12

CARs

Anti-VEGF

VaccineIFN-alphaGM-CSF

Anti-CD40 agonistTLR agonist

ChemotherapyRadiation therapyTargeted therapy

Anti-PD1Anti-PD-L1

IDO inhibitors

Nat Rev Genetics Aug 2016

Combination immunotherapy: More trials, Better targeted

2017 Nature

PD-1/PD-L1 Blockades in MBC

Pembrolizumab in TNBC:Phase Ib KEYNOTE-012 Study

• Response rate: 18.5%

• No response in LDH > 2X UNL

J Clin Oncol. 2016 May 2

Avelumab in Metastatic Breast Cancer

All MBC Patients (N=111)ORR: 3%

TNBC Patients (N=46)ORR: 5%

PD-L1+ PD-L1-

Hyperprogressors!

Clin Cancer Res; 2017

Metastatic Cancer Remission

• Patient selection ?

• Anti-tumor immune response?• How long treat?• Combination approach?• Toxicities?

• Still immune response?• Retreat?

Clinical Questions that you might have….

Immune Targeted Agents

• PD-L1 expression status by IHC: anti–PD-L1 antibody clone 22C3 (Merck)

• Cells counted: neoplastic and intercalated mononuclear inflammatory cells

KEYNOTE-001: Phase Ib study ofPembrolizumab for the Treatment of NSCLC

N Engl J Med 2015;372:2018-28.

Mechanisms of PD-1 Expression

Nature Immunology 14, 1212–1218 (2013)

PD-1/PD-L1: Exhaustion of T cells

1) Antagonizing TCR signaling

2) Decreased survival, proliferation, altered metabolism

3) Reduced proliferation via RAS path

4) Altered transcription

5) Altered T cell motility

Trends in Immunology April 2015, Vol. 36, No. 4

• There are no harmonized criteria to define PD-L1 positivity by IHC1

• Various PD-L1 IHC assays differ in terms of the cells assessed, scoring methods, and thresholds used:

Technical Complexity With PD-L1:Different Thresholds for Expression

NivolumabDako Assay2

PembrolizumabDako Assay3,4

AtezolizumabVentana Assay5,6

DurvalumabVentana Assay7

Cells assessed

Tumour cellsTumour cells and

immune cellsTumour cells and immune cells Tumour cells

Ab clone 28-8 22C3 SP142 SP263

Scoring method

Tumour score: Percentage of cells with membrane staining at any intensity

Proportion score: Percentage of cells with membrane staining at any intensity

Tumour cell (TC) score: staining percentage of tumour cells

Immune cell (IC) score: staining percentage of tumour area

Percentage tumour cells with membrane staining

PD-L1 Expression Levels

<1% or ≥1%, <5% or ≥5%,

<10% or ≥10%≥50%

TC3 or IC3: TC ≥50% or IC ≥10%TC2/3 or IC2/3: TC or IC ≥5%TC1/2/3 or IC1/2/3: TC or IC ≥1%TC0 and IC0: TC and IC <1%

≥25%

Ab, antibody; IHC, immunohistochemistry; PD-L1, programmed death ligand-1. 1. Heskamp S et al. Cancer Res. 2015. [Epub ahead of print]. 2. Phillips T et al. Appl Immunohistochem Mol Morphol. 2015. doi:10.1097/PAI.0000000000000256. 3. Dolled-Filhart M et al. Poster presentation at ASCO 2015. 11065. 4. Rizvi N et al. Poster presentation at ASCO 2015. 8026. 5. Spira AI et al. Oral presentation at ASCO 2015. 8010. 7. Spigel DR et al. Poster presentation at ASCO 2015. 8028. 7. Rebelatto MC et al. Poster presentation at ASCO 2015. 8033.

PD-L1 as a Biomarker?: Issues

• Tumor heterogeneity

• Interval between biopsy and treatment

• Primary vs metastatic disease

• Ab and staining conditions

• Defining a positive result (cut-offs)

- Immune cells vs tumor cells

- Location of expression: intracellular vs surface vs stroma

- Intensity, percent of positive cells

- Distribution

HER-2 type Breast Cancer: PANACEA

2017 SABCS Loi et al

Genomic Signatures of TIL is Prognostic in HER2 Type

2018 BCRT, doi: 10.1007/s10549-017-4502-3

Luminal low Luminal high

HER-2 TNBC

Clinical Development in Cancers with High mutational burden

Science 3 April 2015

Mutational Landscape of Melanoma according to the CTLA-4 blockade Response

Snyder A et al. N Engl J Med 2014;371:2189-2199.

Mutational Load

Survival in Discovery set

Mutation burden, clinical response, and factors contributing to mutation burden: NSCLC

Science 3 April 2015

Neoantigen burden & Intratumoral Heterogeneity (ITH)

Science MAR 2016

Clonal Heterogeneity and Immune Signature in TNBC

N=193 TNBC with follow up data @ TCGAJAMA Oncol. 2017;3(12):1707-1711. doi:10.1001/jamaoncol.2017.2140

TIL Infiltration and Response to anti–PD-1: Melanoma

J Clin Invest. 2016;126(9):3447-3452. doi:10.1172/JCI87324.

Tumoral CD8+ T cell Infiltration after Pembrolizumab

Nature 515, 568–571 (27 November 2014)

Response (n=22)

Progression (n=24)

Proliferating T cells

Change in TCR Repertoire

Genes and Immunity (2016)Nature (2014)

• Theoretical repertoire of 1015–1020 different TCRs that could be generated in humans• Effective anti-tumor immunity increased TCR clonality

T cell Diversity and Mutational Burden

Science 3 April 2015Nat Genetics 2016

What we should know for Rational application of Immune Targeted Agents (ITA)

• Immune responses with standard anti-cancer treatment

• Indicators to judge Immune-related Response

- Pseudo-progression

- Delayed but prolonged response, delayed K-M curve separation

- Mixed responses

• Predictors for Immune-related Adverse Events

• Timing for Re-treatment: monitoring immunosuppressive TME

Best Information is in Tissue

Peter Lee, 2016 SABCS

Multiplex IHC NanoString Analysis: KEYNOTE-001

Assessment of Tumor Immune Microenvironment

Static markers Dynamic markers

• Genomic analysis (WES, targeted seq)• IHC for molecular & immune markers• Flow/CyTOF for phenotyping• RNA seq for profiling the transcriptome• Single cell (TCR seq, RNAseq)

• Analysis of germline SNPs• Flow/CyTOF for phenotyping (best if paired with tumor)• Cytokine profiling in serum• Exosome analysis (WES, RNAseq, paired with tumor)• Single cell (TCRseq, RNAs eq)

Blood mirrors anti-tumor immune responses!

- Immune cell profiles- Cytokines

- PBMC Immunomics

F/81, Metastatic Breast Cancer (TNBC)

Baseline Treatment week #18 Treatment week #16

Cell-based Analysis Flow-cytometry (FACS)

Mass Cytometry (Cy-TOF)

• Easy accessibility• High-throughput• High signal overlap• ~16 parameters

• No signal overlap• >40 parameters• High cost• Low-throughput• Limited availability

MDSC in Ipilimumab Treated Melanoma Patients

Cancer Immunol Immunother (2014) 63:247–257

MDSC with Treatment

MDSC vs LDH MDSC vs M stage

MDSC vs Response

Change in circulating Lymphocytes after Ipilimumab

Early change in ALC Delayed change in CD8

Delayed change in EØ Delayed change in CD4

2016 CCR*Early: 2-8 weeks after Tx, Delayed: 8-18 weeks after Tx.

Anti-PD1 Response: T cell invigoration & Tumor burden

2017 Nature doi:10.1038/nature22079

Anti-PD1 Response: T cell invigoration/Tumor burden ratio

2017 Nature doi:10.1038/nature22079

PennTumor burden (pre) PD-1+CD8 (pre)

MSKCC

Gut microbiome influences efficacy of PD-1–based immunotherapy

Science 05 Jan 2018: DOI: 10.1126/science.aan3706

All cancer

NSCLC

Frequency of

Akkermansia

Dynamic immune monitoring is necessary in

• Development of new IO-based cancer treatment.

: Best combination between ITA-ITA, SOC-ITA.

Endpoint measures

• Decision for treatment, retreatment – Public issue

• Neoantigen discovery & precision medicine

Need collaborative effort in academic society!

Bio-specimens reflecting Anti-tumor Immune Responses

Annu. Rev. Med. 2014.65:185-202

• Translational research for optimal immune monitoring

• Validation of immunoassays using clinical samples

• Establish the most feasible, standardized assays for immune monitoring

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