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

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

FETAL CELLS IN MATERNAL BLOOD- Challenges – are they really there?

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ARCEDI

Are you in there?

FETAL CELLS IN MATERNAL BLOOD- Challenges – are they really there?

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Source Unknown. Couldbe Steen Kølvraa.

FETAL CELLS IN MATERNAL BLOOD- Challenges – are they really there?

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Fetal Cell Type Markers

Rarity of the Fetal Cells

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

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

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

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

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)

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

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

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

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

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

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

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

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

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

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?

2016 – 2017 Operations- Supportive study: BMI - influence upon fetal cell number?

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Starting January 19th, 2017

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

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Until end January 2016 done at BCM

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

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From April 1st part of ARCEDI internal process.

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

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

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