Celvrij DNA - KGBN · Dr. Helena Devos - Hematologie voor de huisarts 2.0 –9 februari 2019 nieuwe...

Dr. Helena Devos - Hematologie voor de huisarts 2.0 – 9 februari 2019

nieuwe diagnostische mogelijkheden

Celvrij DNA:

What is cell free DNA?

Clinical applications:

– Prenatal: NIPT

– In oncology/hematology: Liquid Biopsy

– Population screening for cancer

Content

Cell free DNA (cfDNA)

WHAT?

❖ 1948 cfDNA found in all body fluids

of normal individuals (plasma, urine,

liquor,…)

❖ Small stretches of DNA, about 200 bp

❖ CfDNA amount ↑ if increased cell turn

over (eg. inflammation, sports,…)

❖ 1977: up to 50 times higher in cancer

• Normal + tumor tissue

• Apopstosis (normal cell death)

• Necrosis (due to injury, ischemia, inflammation)

• Active secretion: exosomes: containing cfDNA, circulating RNA, proteins

• Numerical & structural variants, methylation analyse

Cell free DNA (cfDNA)

WHERE DOES IT COMES FROM?

What is cell free DNA?

Clinical applications:

– Prenatal: NIPT

– In oncology/hematology: Liquid Biopsy

– Population screening for cancer

Content

Clinical applications: NIPT

(non invasive prenatal test)

❖1997: cffDNA leaks into the circulation

of the mother

❖CffDNA: placental origin

(syncytiotrophoblast cells)

❖CffDNA: 2-20% of cfDNA in blood

mother~gestational age

❖Rapid decrease of cffDNA after delivery

❖NIPT: targeted (13,18,21,X/Y) versus

whole genome

Heitzer et al. Nature 2017

NIPT: Clinical applications

Generalised mosaic CPM (false+NIPT) CFM (false–NIPT)

❖ From gestation week 12: >4% fetal fraction: NIPT analysis reliable

❖ Screening

❖ ALWAYS confirmation with invasive test

❖ ~1-2% confined placental mosaicism

+16 : low birth weight,

preterm pregnancy

NIPT workflow

❖Streck tube: prevents leakage of DNA into the plasma

❖Plasma cfDNA (< foetus + mother) extraction,

amplification, sequencing, counting of DNA stretches

❖Comparison of amount of DNA for each chromosomal

region with normal foetus

Importance of fetal fractionCalculation of Z-score = degree of deviation from normal(n° reads/average n° reads)Z-score > 3 trisomyZ-score < 3 monosomy

Whole genome NIPT

Abberations in other

chromosomal regions

“side effects”

-from mother or fetus?

-fenotype known?

-ethical questions

• 3/4000 NIPT analysis: maternal aneuploidies

• Presymptomatic cancer diagnosis in these 3 women

Ovarian carcinoma

❖Trisomy screening❖Microdeletion syndromes❖Prenatal RhD genotyping in RhD-mothers❖Other monogenic diseases

Whole genome NIPT:

applications

What is cell free DNA?

Clinical applications:

– Prenatal: NIPT

– In oncology/hematology: Liquid Biopsy

– Population screening for cancer

Content

Liquid Biopsy in Oncology/Haematology

Tissue biopsy (gold standard) CtDNA analysis in plasma

Identifies cancer type + molecular analysis Molecular analysis

Invasive, risk of complications (bleeding, pneumothorax, …)

Painless, non invasive, multiple serial LB possible

Hospitalisation cost Expensive new laboratory techniques, notvalidated yet, research

Limited to one lesion Information about tumor heterogeneity and metastasis

Liquid Biopsy

Laboratory methods

▪ cfDNA: 1ng – 1000 ng/mL

▪ ctDNA = variable (0,01-60% of cfDNA)

▪ Sensitive techniques: dPCR vs NGS

dPCR NGS

targeted abberations panels of genes, wholeexome, whole genome

simple workflow, rapid complex

relatively inexpensive more expensive

highly sensitive less sensitive

CtDNA

Bettegowda et al. Sci Transl Med 2014

Clinical applications of ctDNA

▪ At diagnosis: presence of ctDNA ~ prognosis, can guide treatment

▪ Eg. Melanoma: BRAF V600E mutation (40-60%): sensitive to immunotherapy

▪ Eg. EGFR mutation analysis in NSCLC: excellent response to EGFR-TKIs

▪ In follow up:

▪ Monitoring therapy: presence of residual disease?

▪ Relapse? CtDNA predicts relapse before radiologic progression

▪ Resistant mutations? EGFR T790M resistance mutation - therapy adaptation

CtDNA clinical applicationsin cancer diagnosis

CtDNA analysis

Measurementof total amount

At diagnosis: baseline/prognosis

Follop-up: response to treatment,

MRD

Mutationanalysis

Choice of treatment

Monitoring clonalevolution

What is cell free DNA?

Clinical applications:

– Prenatal: NIPT

– In oncology/hematology: Liquid Biopsy

– Population screening for cancer

Content

Cancer screening: CancerSEEK

Non-metastatic

cancer patients n=1005

Mean sensitivity70%

Cohen et al. Science 2018

Healthy controls n=815

7/815 CancerSEEK +

Specificity> 99%

Plasma analysis: genetic alterations and protein biomarkers -identify the presence of relatively early cancers in 70% -4/5 cases correct localization the organ of origin

Proportion of cancers detected by CancerSEEK (%):

Genomewide copy number alteration screening of circulating plasma DNA: potential

for the detection of incipient tumors

Lenaerts et al. 2018

1002 elderly people

30 aberrant CNA profile

6 (pre)cancerdiagnoses

(hematologic)

24 no cancerdiagnosis

972 normalCNA profiles

4 incidentalcancer

diagnoses

Not yet applicable to the general population: to many FP/FN

Take home message

Clinical applications of cfDNA analysis will grow in the next years

From research to clinics

Applications not only diagnostic, but also prognostic & therapeutic

Thank you

Questions

Take home message

0 10 20 30 40 50 60 70

Liquid biopsy cancer testing

Increased breath of testing (eg. moving from…

Tissue based cancer testing

Companion diagnostic testing

Germline disease testing

Sequencing for pharma

Direct to consumer testing

Other

We do not expect growth of NGS usage

What will drive growth of your NGS usage over the next 2-3 years? (clincal labs N=90, %)

What will drive gowth of your NGS usage over te next 2-3 years? (clincal labs N=90)

Source: William Blair and Genome Web 2017 NGS Survey

Ungoing studies

Promising but

Sensitivity – specificity issues

Sensitive techniques: NGS (=future): parallel sequencing, complex, more expensive, panels, wholeexome , whole genome / ddPCR: targeted, simpleworkflow, rapid, relatively inexpensive, highlysensitive DNA: 30 ng/µL, RNA: 150 ng/µL, proteins,…

NIPT ANALYSIS

FALSE POSITIVE

Vanishing twin

Placental (mosaic) aneuploidie

Maternal deletion or duplication (>20Mb)

Maternal malignancy

FALSE NEGATIVE

Low fetal fraction (high maternal body

weight/BMI, biologicalvariation)

Low fetal mosaic

Sensitive TP53 mutation analysis

NIPT geen diagnostische test, maar gevorderde screening Na afwijkende NIPT is een vruchtwater punctie geindiceerd•Fout-positieven onopgemerkte vanishing twinplacentair (mozaiek) aneuploidie(vervolgdiagnostiek vruchtwater!) maternale deletie of duplicatie (>20 Mb) maternale maligniteit •Fout-negatieven Lage foetale fractie (hoog lichaamsgewicht/BMI, biologische variatie) Laaggradig mozaiek bij de foetus •Test-failure Technische fouten/mislukkingen van afname tot analyse, <3% NIPT niet opnieuw maar invasieve diagnostiek •Onmogelijk Geslachtsbepaling en geslachtschromosomale afwijkingen •Bijvangst whole genome methode vs targetedAneuploidie van andere chromosomen dan 21, 18, 13. Grote deleties, duplicaties

second trimester have been developed.1 The most commonly used

approach is the combination of the NT ultrasound measure at week 12

(week 11-14) and a combination of serum markers assessed using

biochemistry: placental proteins human chorionic gonadotropin (free β,

intact or total) and pregnancy-associated plasma protein-A. etween 70% to over 90% sensitivity at a 5% false positive

rate. For detection of T21, the sensitivity of NIPT is 99.30%

(95%CI: 98.2 to 99.8%) and the specificity is 99.84% (95%CI: 99.69 to

99.92%),

Fig 2. Effect of long term storage of maternal blood (at 22°C) on stability of plasma cfDNA

and cffDNA proportion.

Fernando MR, Jiang C, Krzyzanowski GD, Somer-Shely T, Ryan WL (2018) A novel approach to stabilize fetal cell-free DNA fraction in maternal blood samples for extended period of time. PLOS ONE 13(12): e0208508. https://doi.org/10.1371/journal.pone.0208508https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208508

CfDNA: History - timeline

1948identified in

blood of healthy subjects by Mandel & Metatis

1977: Leon et al. increased amounts of cfDNA found in cancer

patients

2001relating burdenof disease and

cfDNA

Since 2002

Diagnostics Prognostics

KRAS –CRCEGFR-NCCLC

…

1989: Stroun et al

Neoplasticcharacteristics of

ctDNA

1997: Lo et al.cffDNA in

pregnant women (Y- specific DNA)

2011NIPT in

commercial labs

1st line NIPT screening feasablewithout ↑ costs for society?-> Conclusion: Yes if price ↓

RIZIV omzendbrief 1-07-2017

263,65€

False positive NIPT

vanishing twin

placental (mozaic) aneuploidie

maternal deletion of duplication

(>20 Mb)

maternal malignancy

False negative NIPT

Low fetal fraction (high

maternal body weight/BMI,

biological variation)

Low fetal mosaic

Test-failure

Technical errors

Depending on technique (<3% -

>15%)

Invasieve diagnostiek

Cell free DNA (cfDNA): What?

RBC (~5x109 mL/blood)

WBC(~7x106 mL/blood)

CTC (0-10 mL/blood)

PLT (~3x108 mL/blood)

Normal exosomes (~1x1011 mL/blood)

Tumor exosomes (~1x1010 mL/blood)

cfDNA (1ng – 1µg mL/blood)

cfRNA (10ng – 10µg/blood)

Video exosomen???

Exosome formation

DNA

RNA

Proteins

Johann et al. Exp Biol Med 2018

➢ cfDNA (1ng – 1µg mL/blood)

➢ cfRNA (10ng – 10µg/blood)

➢ CTC (0-10 mL/blood)

➢ Normal exosomes

(~1x1011 mL/blood)

➢ Tumor exosomes

(~1x1010 mL/blood)

Cell free DNA (cfDNA)

WHERE DOES IT COMES FROM?

Cell free DNA (cfDNA): Abberations?