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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?
