Carga mutacional tumoral

como biomarcador predictivo

en el contexto de la inmuno-oncología

Eva M Garrido-Martin, PhD

Grupo de Investigación Clínica

en Cancer de Pulmón H12O-CNIO
Disclaimer

• The opinions expressed during this presentation are those of the

speaker and may not represent the opinions of Illumina. Any uses
of Illumina’s products described in this presentation may be uses
that have not been cleared or approved by the FDA or any other
applicable regulatory body.

• Illumina is compensating me to speak at this event.

Non-Small-Cell Lung Cancer

Predictive biomarkers of checkpoint

inhibitors efficacy: PD-L1, TMB

Challenges for implementation of TMB

calculation in clinical practice

Our project
 Lung cancer

One of the most common

cancers (2nd in US)

Leading cause of cancer-

related deaths in US

Poor prognosis
Late onset (>70% advanced)

5 year survival
<50% in early stages

Siegel et al, CA: A Cancer J for Clin 2018

 Non-Small-Cell Lung Cancer (NSCLC)

85% of LC
-ADC
-SCC
-LCC

Chen et al, Nature Reviews Cancer 2014

Genetic aberrations in NSCLC

Modified from TCGA Research Network, Nature 2012 & 2014

 Current NSCLC Therapeutic profile
Early stage – local treatment
• Surgery -> Stages I-IIIA
• Perioperative chemotherapy
• High dose stereotactic body radiation
therapy
• Others (radiofrequency ablation,
standard radiotherapy, chemotherapy)
Advanced stage – systemic treatment
• Thoracic radiotherapy with delivery
of doublet chemotherapy
• Molecular targeted therapies
• Platinum-based doublet therapy
with or without bevacizumab.
• Checkpoint inhibitors
 Timeline of targeted therapies in NSCLC

Genomic
aberrations
discovered

Drugs
Development
(TKIs)

Politi et al, American Association for Cancer Res 2015

 The cancer immunity cycle
Immunotherapy sought-targets in NSCLC

Chen and Mellman, Immunity (review) 2013

Checkpoint inhibitors in immuno-oncology: PD-1/PD-L1
The success of checkpoint inhibitors in NSCLC

KEYNOTE-001

Advanced NSCLC

Garon et al, New Eng J Med 2015

The success of checkpoint inhibitors in NSCLC
CHECKMATE 057
Advanced NSCLC non
squamous that progressed
with chemo-platin

Nivolumab vs docetaxel

Borghaei et al, New Eng J Med 2015

Non-Small-Cell Lung Cancer

Predictive biomarkers of checkpoint

inhibitors efficacy: PD-L1, TMB

Challenges for implementation of TMB

calculation in clinical practice

Our project
PD-L1 as a biomarker in I-O

Garon et al, New Eng J Med 2015

PD-L1 as a biomarker in I-O

KEYNOTE-024

Advanced NSCLC
and PD-L1
expression>50%

Pembrolizumab vs
platinum-based chemo
 Problems in standardization of PD-L1
detection in the routine clinical practice

Blueprint study
n=39

German
harmonization study
n=15

Not all the PD-L1pos patients respond to anti-PD-1/PD-L1

and some PD-L1neg patients are potential responders Hirsch et al, AACR 2016
Scheel et al. Modern Path 2016
Pitfalls of using PD-L1 IHC as a biomarker test for anti PD-1

1. Focal PD-L1 expression in some tumours may be missed in small biopsies

2. PD-L1 expression among multiple tumour lesions in an individual can vary
over time and by anatomical site
3. PD-L1 expression in tumour biopsies collected long time ago might not
accurately reflect PD-L1 status at the moment of treatment, as chemo, radio or
TKIs between collection and immunotherapy can vary its expression
4. PD-L1 epitopes are potentially unstable with fixation and tissue handling
5. Different Ab have different affinities and specificities
6. PD-L1 expression can be membranous (functionally relevant) but also
cytoplasmic
7. PD-L1 can be expressed by multiple cell types with the microenvironment

Topalian et al. Nat Rev Cancer 2016

There is a clear need to find better biomarkers for CPI
From PD-L1 to TMB

Politi et al, American Association for Cancer Res 2015

From PD-L1 to TMB

CHECKMATE-026

Nivolumab was not

associated with significantly
higher PFS or OS than
chemo in patients with
untreated stage IV or
recurrent NSCLC using PD-
L1 as a biomarker
Different cutoff (5%)

BUT

They did observed a

factor that appear in
retrospect to have
had an influence on
the better response of
chemo:
PD-L1 expression
higher in the chemo
group, and
High TMB
 Tumour Mutation Burden (TMB)

TMB is the amount of non-synonym somatic

mutations in the tumour coding DNA relative
to healthy tissue

Increased mutation rate is a well-characterized

feature of human cancer

Abnormal activity in several cellular pathways,

including DNA damage repair and DNA
replication, can increase the overall rate of
somatic mutations in tumours, as can exposure
to mutagens such as ultraviolet light and tobacco
smoke

Defects in DNA damage repair lead to the

accumulation of mutations caused by replicative
errors and environmental damage

• DNA damage / DNA mismatch repair

abnormal activity

• DNA replication abnormal activity

TMB and response rate to PD-1 inhibition

Rosemberg et al, Lancet 2016

Yarchoan et al. NEJM 2017
Tumour Mutation Burden (TMB)

Alexandrov et al, Science 2013

Signatures of mutational processes in human cancer
 Mutational landscape determines
sensitivity to PD-1 blockade in NSCLC

DCB: Durable clinical benefit (partial or stable

response lasting >6 months)
NDB: Non-durable benefit

Rizvi et al, Science 2015

TMB and PD-L1 blockade sensitivity
 Tumour Mutation Burden (TMB):
an emerging biomarker in immuno-oncology

Estimate of the neoantigen repertoire in human cancer

• Aminoacid sequence changes

• Frameshift changes (not included in all TMB
calculations)
• Fusions/rearrangements (not included in all TMB
calculations)
• Structure of the protein recognizable by MHC type I
molecules and therefore, candidate to antigen
presentation

Schumacher et al, Science 2015

Neoantigens in cancer immunotherapy
Neoantigen load has also been correlated with response to
immunotherapy

Van Rooij N, van Buuren MM, Philips D, Velds A, Toeves M, Heemskerk B,

et al. Tumor exome analysis reveals neoantigen-specific T-cell reactivity in
an ipilimumab-responsive melanoma. JCO. 2013;31:e439–42.

However no recurrent neoantigens have been found that predict

response (to date)

Van Allen EM, Miao D, Schilling B, Shukla SA, Blank C, Zimmer L, et al.
Genomic correlates of response to CTLA4 blockade in metastatic
melanoma. Science. 2015;350:207–11.
 Mutational landscape determines
sensitivity to PD-1 blockade in NSCLC

DCB: Durable clinical benefit (partial or stable response lasting >6 months)
NDB: Non-durable benefit

Rizvi et al, Science 2015

TMB and PD-L1 blockade sensitivity
Checkmate 227: TMB as predictor of response with nivo+ipi

Nivolumab anti-PD1
Ipilimumab anti-CTLA-4
Checkmate 227

Hellmann MD et al, (Paz-Ares L) New Eng J Med 2018

Non-Small-Cell Lung Cancer

Predictive biomarkers of checkpoint

inhibitors efficacy: PD-L1, TMB

Challenges for implementation of TMB

calculation in clinical practice

Our project
 How to measure TMB

Targeted panels
300-500 genes
1 – 2 Mb

Whole exome sequencing

WES
180.000 exons
1% human genome - 38 Mb

Whole genome sequencing

WGS
20.000 genes
3,234.83 Mb per haploid genome (x2)

The Cancer Genome Atlas (TCGA) project and several other studies
have used WES to measure TMB across cancer types and found a
wide distribution of TMB across ~20–30 cancer types
Chalmers et al. Genome Medicine (2017)
Challenges for the implementation of TMB
determination in the clinical practice

• Cost

• Sequencing equipment / reference hospitals

• Germline sequencing (tumour-normal pairing)

• Data analysis - Algorithm

• Bioinformatitians

• Servers, storage capacity, optimal computers

• Liquid biopsy

• Turnaround time!!
The Standard of practice: Foundation One Medicine

Comprehensive genomic profiling assay FoM cutoffs:

315 genes TMBlow 1-5 mut/Mb
Exome: 38Mb
1.1 Mb of coding genome TMBmed 5-10 mut/Mb
Correlation to WES: TMBhigh >10 mut/Mb

Chalmers ZR et al, Genome Med 2017

 Available NGS panels for TMB calculation

FDA approved
Horizontal
Panel Brand for diagnostic Technology Genes Bibliography
coverage
use

TruSight Tumour Illumina Hybrid capture

No 170 0,524 Mb
TST170 (DNA+RNA)
AmpliSeq Thermo
Amplicons
Comprehensive Fisher No 409 1,7 Mb
(DNA)
Cancer Panel
Goodman AM,
Mole Canc Ther
Foundation One
Roche Yes Hybrid capture 324 1,1 Mb 2017
CDx
Hellmann MD
NEJM 2018
Gatalica Z EJC
CGP+ Sureselect 2018
ST Caris Molecular Agilent No Hybrid capture 592 1,4 Mb Vandervalde A
Int Cancer Med
2018
Rizvi H et al,
MSK-IMPACT MSKCC Yes Hybrid capture 468 1,22 Mb
JCO 2018
TruSight Oncology
Ilumina No Hybrid capture 523 1,95 Mb
TSO500
Oncomine Tumour Thermo Amplicons
No 409 1,7 Mb
Mutation Load Fisher (DNA)

For Research Use Only. Not for use in diagnostic procedures.

 NGS strategies that I have tried: pros and cons
Ion Ampliseq™ Qiaseq
TruSight®
Comprehensive Comprehensive
Tumor 170
Cancer Panel Cancer Panel

170 genes 275 genes

Genes, Input 409 genes
DNA  RNA DNA  RNA
DNA  RNA
sample and Amplicon-based
Hybridization Hybridization-
capture-based extension-based
Technology
DNA: SNVs,
SNVs, CNVs, CNVs, Indels SNVs, CNVs,
Detected small InDels small Indels
RNA: Fusions,
alterations Splice Variants

1,75 Mb 0,52 Mb 0,83 Mb

Horizontal Mutational burden Mutational Mutational
coverage  burden? burden?

Base Space® Insight Software

Ion Reporter
Data Software
Sequence Hub
Less artefacts
Software Data
analysis More artefacts
Less artefacts (UMIs)

Thermo Illumina Qiagen

For Research Use Only. Not for use in diagnostic procedures.
 Comparison study of technologies using Foundation
One as gold standard

Tissue Illumina AmpliSeq QiaSeq

Patient # TMB MS Mutations Found VUS Total Type of tumour TST170 CCP
origin
ATR, BRIP1, CEBPA, Unknown
D00/MCBM
Med
7/Mb
Stable
STK11, MYC, KEAP1,
TP53
EP300, FAM123B, IRS2,
SDHA, TGFBR2, WT1
13 Brain primary
adenocarcinoma
✓ ✓
ERBB2, NF1, STK11,
Unknown
D01/MEER
High
62/Mb
Stable
ERRFI1, MYC, ASXL1,
CDKN2A, SMARCA4,
44 54 Soft tissue primary
adenocarcinoma
✓ ✓ ✓
TET2, TP53
Unknown
MAP2K1, ARID1A, BRCA2, STAT2, MAP3K1,
D02/ERR
Low
2/Mb
Stable CDKN2A/B, KDM5C, SYK, TNFAIP3, MLL2, 12 Soft tissue
primary
squamous cell
✓ ✓
KDM6A NSD1
carcinoma

D03/MPCR
Low
1/Mb
Stable BCOR, MYB AKT1, AR, CTNNA1, FAT1 6 Lung
Lung adenoid
cystic carcinoma
✓ ✓ ✓
D04/ARA
Low
4/Mb
Stable MLL3 FANCA, WISP 3 Lung
Lung adenoid
cystic carcinoma ✓
Med STK11, CDKN2A/B, Lung
D08/MTLC
15/Mb
Stable
KEAP1, TP53
20 24 Lung
adenocarcinoma ✓
KRAS, PIK3CA, APC, BRIP1, SMAD2, MAP3K1, Unknown
Low
D10/MARP Stable 15 Peritoneum
4/Mb
ARID1A, FAM1234B,
TP53
SMAD3, NOTCH1, ZNF703,
POLE, PTCH1, ROS1
primary
adenocarcinoma ✓

For Research Use Only. Not for use in diagnostic procedures.

 Parameters that accelerate the implementation
of TMB into routine clinical practice

 Manpower

 Expertise (pathology, DNA extraction, library prep, sequencing, data analysis)

 Bioinformatitians

 Open algorithms

 Standardization/Harmonization studies

 Availability of sequencers of high capacity

 Data storage with encrypted data

 Collaboration pathology/oncology/molecular biology

One step beyond: TMB in liquid biopsy

Siravegna G et al, Nat Rev in Clin Oncol 2017

Non-Small-Cell Lung Cancer

Predictive biomarkers of checkpoint

inhibitors efficacy: PD-L1, TMB

Challenges for implementation of TMB

calculation in clinical practice

Our project
Integrating tumour molecular profiling and immune
landscape in NSCLC
 Acknowledgements
Luis Paz-Ares, MD, PhD
Eva Garrido, PhD
Irene Ferrer, PhD
Santiago Ponce, MD
Laura Ojeda
Angela Marrugal
Patricia Yagüe
Javier Ramos
Santiago Garcia
Angel Nuñez
Rocio Suarez
Laura Garcia
Maria Jose Duran
Nuria Carrizo
Mirella Gallego
Estrella Lopez

Rocio Garcia-Carbonero, MD, PhD

Beatriz Soldevilla, PhD
Dr. Ana Belen Enguita Dr. Ivan Martinez Dra. Alicia Maroto Carlos Carretero
Anatomía Patológica Cirugía Torácica Biobanco
Cristina Ojeda
Hospital 12 de Octubre Hospital 12 de Octubre Hospital 12 de Octubre
Beatriz Gil
Dr. Luis Alvarez-Vallina Dra. Lola Martinez
Dr. Daniel Rueda
Inmunoterapia del Citometria de flujo
David Gomez
Cancer CNIO
Tumores Hereditarios
Hospital 12 de Octubre
Hospital 12 de Octubre
Which genomic aberrations do we detect in gDNA and
which do we detect in cDNA?

Type of aberration gDNA cDNA/RNA

frameshift ✓
SNV ✓ ✓
CNV ✓
Small InDels ✓
Large InDels ✓
amplification ✓
rearrangements ✓