Celvrij DNA: nieuwe diagnostische mogelijkheden

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1 Celvrij DNA: nieuwe diagnostische mogelijkheden Dr. Helena Devos - Hematologie voor de huisarts februari 2019

2 Content What is cell free DNA? Clinical applications: Prenatal: NIPT In oncology/hematology: Liquid Biopsy Population screening for cancer

3 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

4 Cell free DNA (cfdna) WHERE DOES IT COMES FROM? 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

5 Content What is cell free DNA? Clinical applications: Prenatal: NIPT In oncology/hematology: Liquid Biopsy Population screening for cancer

6 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

7 NIPT: Clinical applications 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 Generalised mosaic CPM (false+nipt) CFM (false NIPT)

8 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

9 Importance of fetal fraction Calculation of Z-score = degree of deviation from normal (n reads/average n reads) Z-score > 3 trisomy Z-score < 3 monosomy

10 Whole genome NIPT Abberations in other chromosomal regions side effects -from mother or fetus? -fenotype known? -ethical questions

11 3/4000 NIPT analysis: maternal aneuploidies Presymptomatic cancer diagnosis in these 3 women Ovarian carcinoma

12 Whole genome NIPT: applications Trisomy screening Microdeletion syndromes Prenatal RhD genotyping in RhD-mothers Other monogenic diseases

13 Content What is cell free DNA? Clinical applications: Prenatal: NIPT In oncology/hematology: Liquid Biopsy Population screening for cancer

14 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, not validated yet, research Limited to one lesion Information about tumor heterogeneity and metastasis

15 Liquid Biopsy Laboratory methods cfdna: 1ng 1000 ng/ml ctdna = variable (0,01-60% of cfdna) Sensitive techniques: dpcr vs NGS dpcr targeted abberations simple workflow, rapid relatively inexpensive highly sensitive NGS panels of genes, whole exome, whole genome complex more expensive less sensitive

16 Bettegowda et al. Sci Transl Med 2014 CtDNA

17 Clinical applications of ctdna

18 CtDNA clinical applications in cancer diagnosis 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

19 CtDNA analysis Measurement of total amount Mutation analysis At diagnosis: baseline/prognosis Choice of treatment Follop-up: response to treatment, MRD Monitoring clonal evolution

20 Content What is cell free DNA? Clinical applications: Prenatal: NIPT In oncology/hematology: Liquid Biopsy Population screening for cancer

21 Cancer screening: CancerSEEK Non-metastatic cancer patients n=1005 Healthy controls n=815 7/815 CancerSEEK + Mean sensitivity 70% Specificity > 99% Proportion of cancers detected by CancerSEEK (%): 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 Cohen et al. Science 2018

22 Genomewide copy number alteration screening of circulating plasma DNA: potential for the detection of incipient tumors 1002 elderly people 30 aberrant CNA profile 972 normal CNA profiles 6 (pre)cancer diagnoses (hematologic) 24 no cancer diagnosis 4 incidental cancer diagnoses Not yet applicable to the general population: to many FP/FN Lenaerts et al. 2018

23 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

24 Thank you Questions

25 Take home message What will drive growth of your NGS usage over the next 2-3 years? (clincal labs N=90, %) We do not expect growth of NGS usage Other Direct to consumer testing Sequencing for pharma Germline disease testing Companion diagnostic testing Tissue based cancer testing Increased breath of testing (eg. moving from Liquid biopsy cancer testing 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

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29 Ungoing studies Promising but Sensitivity specificity issues

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33 Sensitive techniques: NGS (=future): parallel sequencing, complex, more expensive, panels, whole exome, whole genome / ddpcr: targeted, simple workflow, rapid, relatively inexpensive, highly sensitive DNA: 30 ng/µl, RNA: 150 ng/µl, proteins,

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35 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, biological variation) Low fetal mosaic

36 Sensitive TP53 mutation analysis

37 NIPT geen diagnostische test, maar gevorderde screening Na afwijkende NIPT is een vruchtwater punctie geindiceerd Fout-positieven onopgemerkte vanishing twin placentair (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 targeted Aneuploidie van andere chromosomen dan 21, 18, 13. Grote deleties, duplicaties

38 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: to 99.92%),

39 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): e

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41 CfDNA: History - timeline 1948 identified in blood of healthy subjects by Mandel & Metatis 1989: Stroun et al Neoplastic characteristics of ctdna 2001 relating burden of disease and cfdna Since 2002 Diagnostics Prognostics KRAS CRC EGFR-NCCLC 1977: Leon et al. increased amounts of cfdna found in cancer patients 1997: Lo et al. cffdna in pregnant women (Y- specific DNA) 2011 NIPT in commercial labs

42 1st line NIPT screening feasable without costs for society? -> Conclusion: Yes if price

43 RIZIV omzendbrief ,65

44 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

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46 Cell free DNA (cfdna): What? RBC (~5x10 9 ml/blood) WBC(~7x10 6 ml/blood) CTC (0-10 ml/blood) PLT (~3x10 8 ml/blood) Normal exosomes (~1x10 11 ml/blood) Tumor exosomes (~1x10 10 ml/blood) cfdna (1ng 1µg ml/blood) cfrna (10ng 10µg/blood) Video exosomen???

47 Exosome formation

48 DNA RNA Proteins

49 Cell free DNA (cfdna) WHERE DOES IT COMES FROM? cfdna (1ng 1µg ml/blood) cfrna (10ng 10µg/blood) CTC (0-10 ml/blood) Normal exosomes (~1x10 11 ml/blood) Tumor exosomes (~1x10 10 ml/blood) Johann et al. Exp Biol Med 2018

50 Cell free DNA (cfdna): Abberations?

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