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
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.
<2 weeks
2-8 weeks
>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
istr
ibu
tio
n o
f p
reg
nan
cy l
oss
es
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
rop
ho
bla
st
Yie
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
Tro
ph
ob
las
t Y
ield
(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 Hemoglobinopathies
• Targeted NGS for loci causing inherited disorders
• Sickle Cell Disease (SCD) and Hemoglobin B (HBB)
– Multiplex PCR for loci across HBB exons for NGS
– HbS mutation in HBB gene: GAG GTG (Glu6 to Val6)
– Determine read depth for each allele
• AA Normal Phenotype
• AS Carrier with Normal Phenotype
• SS Affected with SCD Phenotype
– Other loci within HBB for HbC, HbD and HbE variants of SCD
– Variants causing Beta-Thalassemia and Anemia
– Goal: Use targeted NGS to sequence across HBB to genotype fetal DNA
– Verify with Placenta, Newborn Bloodspots, Newborn Medical Records
(HbS)
(HbE) (HbD)
(HbC)
NGS Performance TRIC – Fetal DNA
Maternal DNA
Total number of samples 30 30
Median Coverage depth (idSNPs) 3,607 X 3,521 X
No of called idSNPs (Mean ± SEM) 81 ± 0.4 81 ± 0.4
Median Coverage – HBB loci 12,986 X 19,630 X
No. of HBB loci detected per sample 64 64
idSNPs throughout genome = 90
HBB-specific SNPs = 64
Test Performance for SCD Gene
Accuracy Healthy
Carriers
Confirmed from clinical records 10 8
Confirmed by sequencing corresponding placenta
11 1
By sequencing 21/21 8/9
Performance Parameter Estimation
Sensitivity 89% (8 out of 9)
Specificity 100% (21 out of 21)
Accuracy 97%
Positive Predictive Value (PPV); Precision
100%
Negative Predictive Value (PPV) 96%
F1 score 0.94
Detection of Aneuploidy
• Down Syndrome: Trisomy of chromosome 21
– Collaboration with Susan Klugman, M.D. at Albert Einstein Medical
– Random patients from Wayne State archive
– Patients @Einstein positive for T-21 by NIPT/CVS
– Cervical specimen & Placental tissue obtained at termination
• Fetal and maternal DNA isolated by TRIC
– ForenSeq for determination of Fetal Fraction
– WGA, then Illumina VeriSeqPGS Platform for WGS assay of Chrom. Number
– FISH performed to confirm trisomy
Test Results
• Control Samples (n=18)
– Fetal fraction was >85% in DNA isolated by TRIC (ForenSeq)
– 16 samples Disomic by VeriSeq, with correct gender
• T-21 Suspected Case (collected at 15wk 2d)
– 90% hCG positive cells, 97% fetal fraction in DNA
– VeriSeq: 47/T-21, XY in fetal DNA and placental DNA
– FISH: confirmed fetal T21, D13, D18, XY
• T-21 Suspected Case
– 90% hCG positive cells
– VeriSeq: 47/T-21, XX in fetal DNA and placental DNA
– FISH: confirmed fetal T21, D13, D18, XX
Next-Gen Seq for Chromosome Number VeriSeq PGS & BlueFuse Multi Analysis
Fetus: Male, Diploid Placenta: Male, Diploid Mother
Fetus: Female, T21 Mother Placenta: Female, T21
Co
py N
um
be
r C
op
y N
um
be
r
Chromosome Number
Next-Gen Seq for Chromosome Number N
OR
MA
L
TRIC-Fetus: Male, Diploid Placenta: Male, Diploid
Diploid Aneuploid
Total Samples 16 2
Males 8 1
Females 7 1
Confirmatory Assays
BOBs, Clinical records
BOBs, FISH, Placental material
NIPT
TRIC-Mother
VeriSeq PGS & BlueFuse Multi Analysis
TRIC-Fetus: Female,
T21
TRIC-
Mother
TR
ISO
MY
21
Placenta: Female,
T21
FISH Analysis of Chrs. 13, 18, 21, X, Y
18 Aqua
X Green
Y Red
21 Red
TRIC Isolated Cells
X Green
Y Red
TRIC Excluded Cells
13 Green
21 Red
Exome Sequencing after TRIC
TRIC
REFs
• DNA obtained by TRIC from five pregnancies (S1-S5) with male fetuses
• Compare fetal (F) to maternal (M) cell DNA
• Reference DNA: Newborn blood spots, and maternal blood
Chromosome Coverage
F=Fetal
M=Maternal
5 Weeks
Current Options in Prenatal Testing
15 Weeks 10 Weeks
Amniocentesis CVS NIPT
TRIC
In Summary, TRIC:
• Uses noninvasive cervical sampling
• Isolates hundreds of fetal cells @ >95% purity
• Is effective at 5 weeks of GA
• Unaffected by BMI
• Provides intact fetal cells with complete genome
• Can accurately sequence the fetal genome
• Isolates EVT cells entering the reproductive tract
• Molecular profiling of EVT cells could be useful to identify women at risk for Perinatal Disease
Acknowledgements
Armant / Drewlo Laboratory Team
Leena Kadam
H. Reza Kohan-Ghadr
Brian Kilburn
Nikhil Sarma
Rani Fritz
Anthony Imudia
Alan Bolnick
Jay Bolnick
Chandni Jain
Swati Bajpayee
Nitya Reddy
Department of OB/GYN Clinical Team
Jennifer Smith
Michael Hertz
Javier Rodriguez-Kovacs
Manny Singh
Neil Simmerman
Barbara Crone
Collaborators Marie van Dijk, Amsterdam
Susan Klugman, Bronx
Craig Hartman, Las Vegas
Michael Diamond and
Paul Browne, Univ. Augusta
Berthold Huppertz, Gerit Moser
Graz, Austria
Morey Kraus and PerkinElmer R&D Team, Waltham MA
Gratefully acknowledge the support of:
NIH (HD071408, HL128628, HD092205, HD094405,
HD097904, K12HD001254)
W. K. Kellogg Foundation
PerkinElmer Health Sciences, Inc.
