d. randall armant, ph.d.,cme- for websites... · pdf file current prenatal screening...

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D. Randall Armant, Ph.D., Wayne State University School of Medicine

Trophoblast Retrieval and Isolation from the Cervix

(TRIC)

Disclosures

• Intellectual Property licensed by Wayne State University • Co-ownership of Cradle Genomics, Inc. • Awards from PerkinElmer Health Sciences and NICHD and NIMHD:

• R43HD092205 • R43HD094405 • R43HD097904

http://www.oc2018.cme-congresses.com/ http://www.oc2018.cme-congresses.com/ http://www.oc2018.cme-congresses.com/ http://www.oc2018.cme-congresses.com/ http://www.oc2018.cme-congresses.com/

Brian Kilburn

Trophoblast Retrieval and Isolation from the Cervix (TRIC)

Y

X

Bolnick et. al. (2014) Fertility and Sterility 102:135–142.e6

Trophoblast Migration from the Placenta

• Extravillous trophoblast (EVT) cells – Identified with a specific cell surface antigen – HLA-G

– EVT are disrupted in Perinatal Disease

– Placental cells that reflect Fetal Genome

• EVT cells migrate into the uterine cavity

• Carried by secretion and accumulate in the cervix

• EVT cells are captured by cervical sampling with an FDA approved cytobrush

1. Orr JW, Jr., Barrett JM, Orr PF, Holloway RW, Holimon JL. The efficacy and safety of the cytobrush during pregnancy. 1992.

2. Rivlin ME, Woodliff JM, Bowlin RB, Moore JL, Jr., Martin RW, Grossman JH, 3rd, Morrison JC. Comparison of cytobrush and cotton swab for Papanicolaou

smears in pregnancy. 1993.

3. Paraiso MF, Brady K, Helmchen R, Roat TW. Evaluation of the endocervical Cytobrush and Cervex-Brush in pregnant women. 1994.

4. Foster JC, Smith HL. Use of the Cytobrush for Papanicolaou smear screens in pregnant women. 1996.

5. Holt J, Stiltner L, Jamieson B, Fashner J. Clinical inquiries. Should a nylon brush be used for Pap smears from pregnant women? 2005.

8 weeks

Weeks after

sampling

n=5 n=2 n=1

Safe Non-Invasive Collection from the Cervix

Our Experience Women (N) Median Age

(IQR)

Preg Loss in

1st Trimester

Preg Loss after

1st Trimester

553 25 (22-32.5) 8 12

Percentage 1.5% 2%

National Avg. 9-17% 1% 1920 Ongoing Pregnancies as of November 2017

Published Evidence

0

20

40

60

80

100

1 2 3

% D

is tr

ib u

ti o

n o

f p

re g

n a n

cy l

o ss

e s

Trimester of pregnancy loss

Transcervical Sampling

Anthony Imudia, MD

Endoglandular Trophoblasts

Brown: HLA-G (EVT) Blue: KRT7 (Glands, EVT)

Berthold Huppertz, PhD

Gerit Moser, PhD

Medical University of Graz

From: Moser, Drewlo, Huppertz, Armant (2018) Hum Reprod Update. 24(4):484-496. doi:10.1093/humupd/dmy008

decidua capsularis

decidua basalis

uterine cavity

intervillous space

7 weeks Gestational Age Early First Trimester

11 Weeks

From: Moser, Drewlo, Huppertz, Armant (2018) Hum Reprod Update. 24(4):484-496. doi:10.1093/humupd/dmy008

Current Prenatal Screening Invasive Testing

• Up to 0.5% risk of pregnancy loss in amniocentesis and 1% in CVS

• Invasive, expensive and technically demanding procedure

• Amniocentesis only available later in pregnancy

• Direct sampling of fetal material

• Enables full genetic analysis

• No false results from maternal background

Current Prenatal Screening Noninvasive Prenatal Testing (NIPT)

• Genetic analysis limited to gender, aneuploidy, large deletions (some de novo dominant conditions)

• Mixed sample with dominant maternal background

• Can be impacted by maternal factors

• Not available before fetal heartbeat

• Safe with no risk to fetus

• No complicated medical procedure only blood draw

Trophoblast Retrieval & Isolation from the Cervix (TRIC)

n=224

• Average purity = 96 ± 0.4%

• Avg. yield of HLA-G positive cells =731 ± 31

Fritz et. al. (2015) Prenatal Diagnosis 35:1218-1222

Rani Fritz, DO, PhD

2

4

6

8

10 20 30 40 50 60

ln T

ro p

h o

b la

s t

Y ie

ld

Body Mass Index (BMI)

Advantages of TRIC

• TRIC provides intact fetal cells, entire genome

• Cell recovery is constant with GA & Obesity

• Yield marginally reduced by EPL, IUGR or PE

• Adaptable for High Throughput Automation

Fritz et. al. (2015) Prenatal Diagnosis 35:1218-1222

Obese

2

4

6

8

0 5 10 15 20 25

T ro

p h

o b

la s

t Y

ie ld

( ln

)

Gestational age at sampling (wks)

Slope = -0.169

p value = 0.537

R-sq = 0.002

n = 224

Slope = 0.805

p value = 0.214

R-sq = 0.009

n = 170

Comparisons for Prenatal Genetic Testing

CVS & Amnio NIPT

Safe 99% / 99.5% Yes

Easy (Low Tech) Collection No Yes

Earliest Testing (week) 10 / 15 9 - 10

Fetal Fraction % >99% 2 - 5%

Maternal False Positives No Yes

Maternal False Negatives No Yes

Gender Determination Yes Yes

Aneuploidy Detection Yes Yes

Large Deletions Yes Yes – Limited (>3Mb)

Monogenic Disorders Yes No

Clinically Relevant CNVs Yes No

Whole Genome Seq Yes No

Pregnancy Indicators No No

TRIC

Yes

Yes

5

85 - 99%

No

No

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Leveraging TRIC TRIC

De-/Trans-Differentiation

Placentology

Risk-assessed tissues EVT fr. TRIC/placentas

EVT Culture Isolate Live EVT

Cell Lines

Obstetric Disorders

Biomarker Discovery:

Transcriptomics

Proteomics

Epigenomics

Metabolomics

Prenatal Genetics

Single Gene Disorders

Aneuploidies

Whole Genome Sequencing

Whole Exome Sequencing

DNA Methylation

TRIC Potential for Prenatal Genetic Testing

• Approx. 600pg gDNA/100 cells • Complete genome is present • Next-Generation Sequencing (NGS) • Whole Genome/Exome Sequencing

Collaborator

Dr. Sascha Drewlo

Red = X chromosome Green = Y chromosome

FISH:

Nuclei Isolate from

Male Fetuses by

TRIC

Fetal v/s Maternal Fetal v/s Placenta

Preparation of Fetal DNA after TRIC

F-Actin Nuclei

100 𝝁m

Intact cells on slide Cytoplasm removed Immobilized DNase Treatment

Retained Nuclei

Targeted NGS analysis: Maternal and Fetal DNA contained identical haplotypes across all SNPs Conclusion: Isolated fetal cells contain maternal DNA fragments Solution: Isolate Nuclear DNA before NGS

Jain et al. (2016) Science Transl. Med. 8, 363re4

Preliminary Experiment

Lysis and DNA purification

Experimental Design n=20 Triads: Fetal Cells obtained by TRIC Maternal Cells obtained by TRIC Placental Villi after termination Informative M and F STRs/SNPs compared: Fetal Fraction Determined

59 STRs 94 SNPs

Jain et al. (2016) Science Transl. Med. 8, 363re4

Maternal vs Fetal

Placenta vs Maternal

Placenta vs Fetal

Analysis of Fetal DNA by NGS after TRIC

STR Analysis: SNP Analysis:

Jain et al. (2016) Science Transl. Med. 8, 363re4

High Purity – High Fetal Fraction Targeted NGS of 20 TRIC/ Maternal/Placental Triads

Gestational

Age (weeks)

Trophoblast cell purity

(% β-hCG)

Fetal

Gender % Fetal

Fraction

5 86 M 97.6

5 89 F 92.2

5 91 F 87.2

6 85 F 95.2

6 92 M 85.6

6 93 M 75.6

6 89 M 93.9

6 86 F 98.6

6 89 M 100

7 92 F 82.1

8 80 F 82.4

8 96 F 97.2

8 96 F 90.8

8 92 F 92.8

9 85 M 92.9

9 94 M 100

11 85 F 95.9

13 91 M 91.9

14 90 M 92.6

19 80 F 98.7

Average: 8.3 ± 3.6 Weeks

Average: 89.2 ± 5.0%

Average: 9-M; 11-F

Average:

92.2 ± 6.5% Jain et al. (2016) Science Transl. Med. 8, 363re4

Reza Kohan-Ghadr,

PhD

Leena Kadam, PhD

Fetal DNA Obtained by TRIC

• Rigorous DNA interrogation by targeted NGS

• Fetal haplotypes matched placenta references

• Fetal haplotypes distinct from maternal DNA

• All 59 STR and 94 SNP sites correctly called in every sample

Jain et al. (2016) Science Transl. Med. 8, 363re4

Prenatal Test for Single Gene Diseases Hemoglobino