cell based nipt (cbnipt) extravillous trophoblasts as...
TRANSCRIPT
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Cell-based NIPTJP Morgan January 2017
Palle Schelde CEO
ARCEDI BiotechDenmark
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ARCEDI METHOD - OVERVIEW- Enrichment, Detection and Analyses of Circulating Fetal Cells
Scanning and
Identification of Fetal
Cells
Selection
and Staining
using
ARCEDI
markers
Pregnant
women
GA: 10 to
13 weeks
Blood
Processing
(30mL of
whole
blood)
Positively
Identified Fetal Cell
‘Picking’ the
Fetal Cell
Whole Genome
Amplification
(WGA)
CBNIPT
using
CMA/NGS 2
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CHECKLIST- Technology to be approved as a cbNIPT/D should fulfil the following criteria
IDENTIFICATION OF FETAL CELL TYPE
MARKERS SENSITIVE AND SPECIFIC FOR FETAL CELLS
ACCESSIBILITY OF FETAL CELLS FOR DOWNSTREAM ANALYSIS
VIABILITY OF FETAL CELLS FOR PRENATAL TESTING/DIAGNOSIS
ROBUST METHOD TO ENRICH AND IDENTIFY FETAL CELLS
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FETAL CELLS IN MATERNAL BLOOD- Challenges
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Beginning Cb-NIPT Enrichment from MB
Skepticism
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FETAL CELLS IN MATERNAL BLOOD- Challenges – are they really there?
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ARCEDI
Are you in there?
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FETAL CELLS IN MATERNAL BLOOD- Challenges – are they really there?
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Source Unknown. Couldbe Steen Kølvraa.
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FETAL CELLS IN MATERNAL BLOOD- Challenges – are they really there?
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Fetal Cell Type Markers
Rarity of the Fetal Cells
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FETAL CELL TYPE- Establishing the Fetal Cell gene expression profile
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• Positive selection using 4 different endothelial/MSC markes
• Positive selection using CD105
198FC
2 PIQOR STEM CELL ARRAY
39 39 36
- 22 Genes expressed in Placenta- Out of these 13 Genes expressed in
Extravillous Trophoblasts
Array 1 Array 2
FETAL CELLS ARE OF PLACENTAL ORIGIN
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FETAL CELL TYPE- Establishing the Fetal Cell gene expression profile
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FETAL CELL TYPE- Trophoblast mediated Uterine Vessel Remodelling
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• EMT (Epithelial – Mesenchymal Transition)
• Fetal Cells that shed in the Maternal Circulation express markers for both:
• EPITHELIAL CELLS
• ENDOTHELIAL CELLS
• ARCEDI MARKER COMBINATION!
• 8 Markers for Enrichment and Staining of Fetal Cells
Moser et al. 2016
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FETAL CELL TYPE- Establishing the Fetal Cell gene expression profile
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ARCEDI METHOD – MARKER SPECIFICITY- Misclassification of MCs as FCs
• 143 FETAL CELLS FROM MALE PREGNANCIES.• After identification, Fetal Cells subjected to XY FISH
XY 116
XX 0
No. FISH Signal 19
Lost during FISH 8
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FETAL CELL TYPE- Establishing the Fetal Cell gene expression profile
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ARCEDI METHOD - MARKER SENSITIVITY- Method optimization 2014 - 2016
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STUDY 1
2014 (Hatt et al.)
STUDY 2
2016
No of Samples 78 178
No. of Scanning images 5000 350
Average fetal cells 4.3 12.5
Range fetal cells 0-18 1-45
% no calls (zero fetal cells) 9 0
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ARCEDI METHOD – MARKER SENSITIVITY- Performance - Retrieval of Fetal Cells
• 178 PREGNANT WOMEN at NT SCAN- 30ml Blood.• 89 SAMPLES FROM ‘LOW RISK’ GROUP
• 89 SAMPLES FROM ‘HIGH RISK’ GROUP (offered CVS)
PARAMETER VALUE
No of Fetal Cells 2227
Mean (per sample) 12.5/30ml blood
Median 10
Range 1-45
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ARCEDI METHOD – MARKER SENSITIVITY- Frequency Distribution (‘High Risk’ vs ‘Low Risk’)
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ARCEDI METHOD – ROBUSTNESS- Sample Stability (Parameter/Platform Independant)
Fetal Cell Number and Distribution unaffected by:
• Blood collection tubes (BD (EDTA tubes) vs Streck Tubes)
• Time before blood processed – 24 hrs and 48 hrs - currently investigating 5 days
• Transportation – air and road and processed after 48 hrs
• Fetal cells from every sample!
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ARCEDI METHOD – VIABILITY- Decifering Genetic Information from Fetal Cells
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ARCEDI METHOD – VIABILITY- Case III (NIPT413): Mosaicism [45,X/46,X,r(X)] – array CGH
Array CGH on FC
Array CGH on Amniocentesis
377 450
377
450
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Kølvraa et al. 2016(Prenatal Diagnosis)
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ARCEDI METHOD – VIABILITY- Case III (NIPT413): Mosaicism [45,X/46,X,r(X)] - NGS
45,X: cell 377
46,X,r(X): cell 450
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2016 -2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
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Pregnancies
(6 Hospitals in DK)
CVSFetal Cells from maternal blood
Fetal DNA from maternal plasma
• Recruit ‘High Risk’ pregnancies (which areoffered and accept CVS) from 6 different hospitals in Denmark. Current enrollment 64 pregnancies.
• Isolate Fetal Cells from all blood samples
• Perform cell based fetal DNA analysis on selected samples
• Perform cell free fetal DNA analysis from the plasma
• Check whether the results from all the three tests correlate
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2016 -2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
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Case HR235-1:GA: 13+4Risk assessment: T21 1:6891; β-hCG elevated.
5 fetal cells wetpicked
Case HR235-1:Gender determination: male fetusGenes testet: DYS 14 (TSPY 1) and SRY
a-CGH (Agilent 180K) result from CVS a-CGH (Agilent 180K) result from fetal cells
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2016 -2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
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Case HR240-2:GA: 13+2Risk assessment: T21 1:237
4 fetal cells wetpicked
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2016 -2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
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Case H248-1:GA: 12+3Risk assessment: T21 1:17
3 fetal cells wetpicked
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2016 -2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
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Case H255-1:GA: 13+3Ultrasound revealed:Dichoronic twinsRisk assessment: T1 (F);T21 1:12000
T2 (M); T21 1:36
1double fetal cell wetpicked
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2016 -2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
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Case H256-1:GA: 13+5Risk assessment: T21 1:133
3 fetal cells wetpicked
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2016 -2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
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Case HO3-1:GA: NARisk assessment: Parents have known inv chr. 6
3 fetal cells wetpicked
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2016 -2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
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Case 1991-1:GA: 12+4Risk assessment: Low riskGender: XX
4 fetal cells picked from a MMI slide
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2016 – 2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
Case HR146-3:GA: 12+4
Risk assessment: T21 1:2
CVS DNA: Q-STR confirmative for 4 out of 6 markers on chr 21 – inconclusive for 2. Confirmative for T21. No a- CGH analysis.
7 fetal cells dry picked
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2016 – 2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
Case HR146-3:GA: 12+4
Risk assessment: T21 1:2
CVS DNA: a- CGH result
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2016 – 2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
47,XY,+21HR146-1: 7 cells
HR146-1: Male Pregnancy (Trisomy 21; Risk 1:2; maternal age 37; NT 5,3)
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2016 – 2017 Operations- Study (CVS vs cffNIPT vs cbNIPT)
ARCEDI NO GA Risk assessment CVS NIPT (Verify - Illumina) no FCs cbNIPT Q-PCR notes
HR146 12+4 T21 1:2 47 XY (T21) NA 7 47 XY (T21) XY
QST*R: 4 out of 6 markers for chromosome 21 showed 3 allels, 2 markers were inconclusive
HR235 13+4T21 1: 6891 beta hCG 7,1 MoM 46 XY
Normal result reported for 21, 13,18, X and Y. 5 46 XY XY
HR240 13+2 T21 1: 237 46 XXNormal result reported for 21, 13,18, X and Y. 4 46 XX XX
hr248 12+3 T21 1: 17 46 XXNormal result reported for 21, 13,18, X and Y. 3 46 XX XX
HR255 13+3TV1: T21 1: 12000 TV2: T21 1: 36
TV1: 46 XX TV2:46 XY
Normal result reported for 21, 13,18, X and Y. 2 (1 double cell) 46 XY
Presence of Y
material Dichoronic twins
HR256 13+5 T21 1:133 46 XXNormal result reported for 21, 13,18, X and Y. 3 46 XX XX
HO3 NAKnown inversion chromosome 6 46 XY NA 5 46 XY XY
1911 12+4 None (low risk) NA NA 4 46XX XX MMI slide
In total: 72 samples banked. 30+ used for reagent optimization (dry picking - wet picking). Current pool no del/dup.
Current back lock for a-CGH: 12 samples. 2 NIPT no-calls due to low fetal fraction (maternal BMI above 30). Offered CVS.
Currently: 100% correlation betwen CVS/cffNIPT/cbNIPT
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CHECKLIST- Technology to be approved as a cbNIPT/D should fulfil the following criteria
IDENTIFICATION OF FETAL CELL TYPE
MARKERS SENSITIVE AND SPECIFIC FOR FETAL CELLS
ACCESSIBILITY OF FETAL CELLS FOR DOWNSTREAM ANALYSIS
VIABILITY OF FETAL CELLS FOR PRENATAL TESTING/DIAGNOSIS
ROBUST METHOD TO ENRICH AND IDENTIFY FETAL CELLS
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2016 - 2017 Operations- Supportive study: Fetal Cell Variation with Gestational Age
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Scope of study:• investigate fetal cell variation with gestational age
Inclusion criteria:• 10 study individuals gestational age week 8; 12 and 18 (same individuals)
Study Collaborator:• Virtus IVF Clinic, Skejby (Private Clinic), Denmark
Study lay out:• Bloodsampling, processing and counting of fetal cells at each of the GA points
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2
13
3
4
8
2016 - 2017 Operations- Supportive study: Fetal Cell Variation with Gestational Age
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Scope of study:• investigate fetal cell number post delivery
Inclusion criteria:• Group 1: 10 study individuals having delivered within the past 48 hours. No knowledge of previous
pregnancies. Individuals will be followed at 14 days and 30 days.
• Group 2: 10 study individuals having delivered within the past 48 hours. Knowledge of previous firstpregnancy being a male and current pregnancy being a girl. Individuals will be followed at 14 days and 30 days.
Study Collaborator:• Dept. for Womens Diseases and Births, Aarhus University Hospital, Denmark
Study lay out:• Group 1: Bloodsampling, processing and identification of fetal cells.• Group 2: Bloodsampling, processing and identification of fetal cells. Verification by FISH.
2016 - 2017 Operations- Supportive study: Fetal Cell persistence after delivery
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2016 - 2017 Operations- Supportive study: Fetal Cell persistence after delivery
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Supportive study: fetal cell persistence after delivery
Study Group Individual no Time point Number of fetal cells
1 1 48 hours 7
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Scope of study:• investigate fetal cell number from pregnancies having a BMI of 30 or above
Inclusion criteria:• Individuals having a BMI of 30 or above.
Study Collaborator:• Dept. for Womens Diseases and Births, Aarhus University Hospital, Denmark
Study lay out:• Group 1: Bloodsampling, processing, identification and counting of fetal cells. All individuals will be
offered NIPT. For those individuals being grouped as high risk also CVS will be offered.
2016 - 2017 Operations- Supportive study: BMI - influence upon fetal cell number?
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2016 – 2017 Operations- Supportive study: BMI - influence upon fetal cell number?
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Starting January 19th, 2017
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ARCEDI METHOD - Turnaround time/sample
0 1 5 12 12.511
ARCEDI method: Processing Time per sample in Hours (continuous)
Sampling BloodProcessing
FetalCellEnrichment and
staining
Smearing and drying
Mounting andscanning
Visual inspection Picking
13.5 20.5
WGA
40
Until end January 2016 done at BCM
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ARCEDI METHOD - Turnaround time/sample
0 1 5
ARCEDI method: Processing Time per sample in Hours (continuous)
Sampling BloodProcessing
FetalCellEnrichment and
staining
Mounting andscanning
Visual inspection Picking
15.5
WGA
41
8
From April 1st part of ARCEDI internal process.
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ARCEDI METHOD - Instrumentation
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Diagnostic options:
• Genotyping each cell using STR or SNP’s
• Array CGH (CMA) for CNV’s
• NGS for detection of CNV’s
• NGS for detection of point mutations
Blood processing Fetal cell enrichment and staining Scanning, Validation and Cell picking Whole Genome Amplification
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Patent
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Patent
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Patent
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Patent
Latest granted areas:
Japan and Eurasia
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Fetal Cells in Maternal Blood- Who is active in the field?
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FETAL CELL TARGETTED APPROACH1
KNOWLEDGEOF CELL TYPE2
BIOLOGY DRIVEN3
FETAL CELL MARKERS4
DNA FIDELITY5 CLINICALVALIDATION6
PUBLICATONS7
RARECELLS(filter separation)
FETOLUMINA(immobilized Ab)
KELLBENX(NRBC’s; 4b9 Ab)
RARECYTE(float tube sep)
SILICON BIOS.(DepArray)
ARCEDI(specific Ab comb.)
1 Whether the technology was developed for a different rare cell type (CTC) or it was targetted for fetal cells right from the beginning2 Whether the technology is developed on the basis of a specific cell type3 Whether the technology is driven by biology or instrumentation4 Whether there is knowledge of fetal cell specific markers5 Whether enriched fetal cells have been tested for the quality and quantity of DNA6 Whether the technology has gone through clinical validation 7 Whether the technology has been peer-reviewed
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THE TEAM……AND BEYOND
ARCEDI Biotech• RIPUDAMAN• LOTTE• KATARINA• PETER• SIMON• MATHIAS• SOFIE• ANNE• MICHEL• MAIKEN• FILIZ• HAI QING• RIE• TINE• RIKKE
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AUH (Dept. Gyn/Obs)• NIELS• OLAV• MARIANNE• LOTTE• ALICE
AUH (Dept. Clinical Genetics)• IDA• ELSE MARIE• RIKKE