improving cancer therapy through molecular diagnostics...

Improving cancer therapy through molecular

diagnostics

Nestebiopsia ja

solunulkoinen DNA

syövän merkkiaineena

Juha Kononen MD PhD

Conflict of interest statement

❖ Received honoraria for lectures from Amgen, Roche,

AstraZeneca, Celgene, Glaxo-Smith-Kline, Lilly

❖ Participated in international oncology meetings with

support from Roche, Novartis, Amgen, Celgene

❖ Participated in advisory board meetings for Celgene,

GSK, Roche, BMS, Pfizer

Liquid biopsy: topics

• Why:

✴ Need for molecular diagnostics

✴ Importance of follow-up and re-testing

• How:

✴ Methods

✴ Opportunities, requirements, limitations

• Examples

Paradigm: oncogene addiction

❖ Implications: Driver

mutations

❖ “Precision medicine”

❖ Predictive

biomarkers

❖ Tissue is the issue:

quality, availability,

representativity

Need for re-testing: clonal evolution during

treatment

Tumors are heterogeneous

Cells that have growth advantage form bulk of the metastatic lesion

No two cancer cell genomes are

identical

Wang et al., Nature 2014

Driver mutations in single cancer cells (TNBC)Molecular evolution rate.

Mutation frequency is 13x higher in TNBC compared to ER+ cells

Biological systems have evolved to sense and

integrate noisy signals from environment

❖ Feedback-loops

❖ Multiple

homologous growth

factor receptor

families

❖ Parallel signalling

cascades

Cancer evolves.

Surgical biopsies are not always

feasible.

Metastatic lesions are heterogeneous.

How to dynamically monitor which clone

forms the bulk of disease burden?

Liquid biopsy:

Tumor-derived

nucleic acids are

present in blood.

Sources:

circulating

cancer cells,

cell-free DNA

Exosomes in cancer

Liquid biopsy for monitoring clonal selection and

molecular evolution

ddPCR is a sensitive method for analyzing known driver mutations from plasma samples

Measuring molecular residual disease

❖ TAYS/KSSHP adjuvant

study

❖ 80 stage III CRC cases

❖ Liquid biopsy follow up

for 3 years after surgery

2. line

Liquid biopsy may predict tumor progression

Misale S, et al. Nature 2012;486:532‒536

Diaz LA, et al. Nature 2012;486;537‒540

Vilar E, Tabernero J. Nature 2012;486:482‒483

Anti-EGFR treatment

KRAS-mutant ctDNA

Other mutant ctDNA

0 4 8 12 16 20 24

Weeks

ctD

NA

am

ount

SD with imagingRadiological disease

progression

Liquid biopsy

Tumor

Metastasis

0 4 8 12 16 20 24

Implications for treatment: re-challenge

❖ Molecular makeup

of bulk of tumor

may fluctuate

during treatment.

❖ Cancer may be

sensitive for

intermittent

treatment.

Siravegna, G. et al. (2015). Clonal evolution and resistance to EGFR blockade in

the blood of colorectal cancer patients. Nature Medicine, 21(7), 795–801.

RAS-määritysmenetelmien herkkyys

Normanno N, Pinto C, Castiglione F, Bardelli A, et al. (2011) KRAS Mutations Testing in Colorectal Carcinoma Patients in Italy: From Guidelines to External Quality Assessment. PLoS ONE 6(12): e29146. doi:10.1371/journal.pone.0029146

Method Sensitivity, % total DNA

Sanger Sequencing 10-25

PCR / RFLP 10

Pyrosequencing 2-10

NGS 2-10

ARMS/Scorpion probes (Therascreen) 1

BEAMing 0.1-1

COLD-PCR 0.1-1

digital droplet PCR 0.01-0.1

Techniques used at KSSHP: Pyrosequencing, NGS, ARMS/Scorpion probes, e-ICE-COLD-PCR, ddPCR

ddPCR = Droplet Digital PCR

Droplet

digital PCR

• Quantitative

• Sensitive

• Suitable for both

mutation and copy

number analyses

• Can be combined with

other PCR methods

Basic concepts: sample partition

• Reactions performed and analysed in each droplet separately

Creating and reading droplets

• Mixing oil with samples

creates uniform reaction

droplets

• Individual droplets are separated

for oil with samples creates

uniform reaction droplets

• Droplets are transferred

to 96 well plate for PCR

Measuring and quantification

• Fluorescent readings

measured for each

droplet in two channels

• Droplets are assigned

as positive or

negative by

thresholding based

on fluorescence

amplitude.

• Count of positive and

negative droplets and

poisson-based 95 %

confidence intervals

are calculated.

Case example:

• Originally response to first-generation TKI

• Mixed response in control CT and some metastatic

lesions showing progression during treatment.

• Analysis of EGFR mutation status from plasma

• Plasma sample preparation in Pori. Frozen plasma

shipped to Jyväskylä for nucleic acid extraction and

ddPCR analysis

Exon 19 del

• Frequency: approximately 18

%

EGFR

p.E746_A750del

WT

p.E746_A750d

el + WT

T790M mutation

• Frequency of mutation: approximately 5 %

• Heterogeneous tutor burden: both sensitive and resistance mutation detected from plasma

• Interpretation: selection of resistant clone during treatment -> switch to another TKI

Liquid biopsy at KSSHP

• EGFR testing: exon 19 del, L858R, T7980M

• RAS mutation: KRAS and NRAS mutations

• BRAF V600

• PIK3CA

• Her-2 amplification

• EGFR copy number analysis

• Custom assay development for any mutation

Tutkimuspyyntö 8000 P-EGFR-D

EGFR-geenin mutaatiotutkimus, plasma

• 9 ml plasmaa

• EDTA putket, erottelu mahdollisimman pian (<2h)

• Näytteen mukaan erillinen lähete (taustatiedot, kysymyksenasettelu)

• Sairaalan sisältä plasma viiveettä molekyylipatologian laboratorioon

• Ulkopuolelta tulevat näytteet lähetetään pakastettuna ja kuivajäissä

• Menetelmä: DNA-eristys ja alleelispesifinen qPCR tai ddPCR

• Tulkinta: Kirjallinen lausunto

• Aika: n. 2 viikkoa

• Hinta: 430 eur

QC

• The European Society of Pathology(ESP) established an EQA program for

testing biomarker mutations in non-small cell lung carcinoma (NSCLC). This

program aims to ensure optimal accuracy and proficiency in lung cancer

biomarker testing across all countries.

• The practical organization of this European EQA program is done in

collaboration with the members of the ESP Lung EQA scheme steering

committee and the Biomedical Quality Assurance Research Unit of the KU

Leuven, lead by Prof. Dr. E Dequeker. The ESP Lung EQA program works in

close contact with Prof. Dr. H van Krieken, president of the ESP. The scheme

is supported by an educational grant from Pfizer.

• This scheme is in collaboration with UK NEQAS ICC&ISH.

• The ESP EQA schemes are accredited by BELAC conform the ISO 17043

QC results 2016

• Osallistujia eri maista 114 laboratoriota

• 59 laboratoriota (52 %) sai hyväksynnän

• KSSHP: 17,5 / 18 pistettä - kirkkaasti hyväksytty

Drug response and sensitivity testing

New approach to cancer therapy

Living biobanks

Drug sensitivity testing

Genome analysis

Immunophenotypes

Transcription profiling

Tumor

Biopsy

Dissociation

Cell sorting

Monitoring molecular

residual disease with serial liquid

biopsies

Diagnostic histology

Experimental

protocols

Response

evaluation

Predictive and

personalised biomarker

development

Adjust therapy

accordingly

Lung cancer in

a non-smoker

Liquid biopsy

NGS

Standard therapyLiquid biopsy

ddPCR

Acknowledgements

Ismo Jantunen

Teijo Kuopio

Aleksi Isomursu

Laura Lahtinen

Noora Nykänen

Outi Välilehto

Reino Pitkänen

Kaija Vasala

Vesa Kataja

Juha Rantala

Rami MäkeläCaroline Heckman

Henrik Edgren

Samuli Eldfors

Astrid Murumägi

Maija Wolf

Olli Kallioniemi

Kimmo Porkka

Erkki Elonen

Kaisa Lehtomäki

Tapio Salminen

Juhani Sand

Kaisa Sunela

Leena Keskinen

Ismo Strander