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

Click here to load reader

Post on 14-Oct-2020

0 views

Category:

Documents

0 download

Embed Size (px)

TRANSCRIPT

  • www.cogen2018.cme-congresses.com

    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