NIPT Market & Tech Analysis Report 2015 - Li MA

Market & Tech analysis report

Non-Invasive Prenatal Testing

Produced by Li Ma, Msc, PhD candidate 2015

NIPT (Non-Invasive Prenatal Testing)

什么是无创产前检测 (NIPT) ?通过采集孕妇的外周血，提取游离 DNA (cell free DNA) ，从而安全准确的检测常见的胎儿非整倍性染色体异常。- 唐氏综合征（ T21 ）- 爱德华氏综合征（ T18 ）- 帕陶氏综合征（ T13 ）

Incidence of common chromosome abnormalities

发病原因 疾病名称 发病率

21 号染色体三体 唐氏综合症Down Sydrome 1 ： 800

18 号染色体三体 爱德华氏综合症Edward Syndrome 1 : 3500 – 8000

13 号染色体三体 帕陶氏综合症Patau Syndrome

1 : 25 000

Facts about Down Syndrome

年龄 发病率 < 35 < 0.3 %

37 0.5 %40 1 %50 10 %

- 最常见的染色体非整倍体疾病 (1:800)- 先天性智力低下，生长发育迟缓，并伴发多系统畸形- 随年龄增加发病率增加， >35 岁直线上升

Screening and diagnosis of DS at clinical setting

94%-96% 5%-6%

Screening and diagnosis of DS at clinical setting

无创产前筛查 (NIPT)

1997 : Lo et al. : cff DNA in maternal circulation

10-20 % of cell-free DNA in maternal circulation is fetal in origin

无创产前筛查 (NIPT)

Shotgun Massively Parallel Sequencing (s-MPS) Gregg, A. R., et al. (2014). Annu Rev Genomics Hum Genet

Importance of fetal fraction

Fetal Fraction

Expected ratio for Trisomy

4% 1.02

10% 1.05

20% 1.10

40% 1.20

Shotgun Massively Parallel Sequencing (s-MPS)

Shotgun Massively Parallel Sequencing (s-MPS) Z-score

Distribution of %ChrNreference

%ChrNreference

Mean %ChrNreference

euploid T21

3σ

Z-score < 3 Z-score > 3

Shotgun Massively Parallel Sequencing (s-MPS) Z-score

Case-control study including 1696 pregnancies Detection rate (Sensitivity) – 98.6% (209/212) False-positive rate: 0.2% (3/1471)

0.8% risk of test failure

Shotgun Massively Parallel Sequencing (s-MPS) Z-score

0.3 million (SD 88,000) reads 2.3 million (SD 474,000) reads

Shotgun Massively Parallel Sequencing (s-MPS) Importance of read depth

Shotgun Massively Parallel Sequencing (s-MPS) Importance of read depth

• Chr13 and Chr18 have relatively lower average GC content than Chr21 which has a modest average GC content.

chr13 chr18

SBS

SBL

chr21

1

1

5.8X 2.4X

5.7X 1.8X

• Coefficient of variation (CV) - Genomic representations of chr13 and chr18.

Z-score with GC bias correctionShotgun Massively Parallel Sequencing (s-MPS)

Z-score with GC bias correctionShotgun Massively Parallel Sequencing (s-MPS)

Trisomy 18

Detection rate

False positive rate

Trisomy 13Trisomy 21

100% (59/59)

0.3% (5/1,688)

91.7% (11/12)

0.9% (16/1,688)

99.1% (210/212)

0.1% (1/1,688)

Z-score with GC bias correctionShotgun Massively Parallel Sequencing (s-MPS)

The count of mapped sites for a given chromosome of interest is normalized to counts observed on another pre-determined chromosome (or set of chromosomes).share similar GC content

Normalized Chromosome Value (NCV)Shotgun Massively Parallel Sequencing (s-MPS)

Chr21/Chr1 Reference MeanChr 21/Chr1 Sample

Amy J. Sehnert, 2011

Chr21/Chr1 Reference SD

Normalized Chromosome Value (NCV)Shotgun Massively Parallel Sequencing (s-MPS)

Case control study including 532 pregnancies. Detection rate (Sensitivity) for T21 – 100% (89/89), No false-positive (404/404).

2.6% unclassified (NCV between 2.5 and 4).

Detection rate (Sensitivity) for T13 – 78.6% (11/14), No false-positive (485/485). Detection rate (Sensitivity) for 45,X – 93.8% (15/16), Specificity – 99.8%

(416/417).

Detection rate (Sensitivity) for T18 – 97.2% (35/36) , No false-positive (460/460).

Normalized Chromosome Value (NCV)Shotgun Massively Parallel Sequencing (s-MPS)

Targeted Massively Parallel Sequencing (t-MPS)DANSE assay, FORTE algorithm

Left probe Middle probe Right probe

Polymorphic site

Determine fetal fraction

Non-Polymorphic site

Determine copy number

Targeted Massively Parallel Sequencing (t-MPS)DANSE assay, FORTE algorithm

Trisomic model

Disomic model

Observed proportion for sample j

Estimated reference proportion

Fetal fraction

The FORTE algorithm estimates the risk of aneuploidy using an odds ratio comparing a model assuming a disomic fetal chromosome and a model assuming a trisomic fetal chromosome.

Targeted Massively Parallel Sequencing (t-MPS)DANSE assay, FORTE algorithm

Case control study involving 400 pregnancies. Detection rate (Sensitivity) for T21 – 100% (50/50), specificity - 100% (297/297).

1% test failure.

Detection rate (Sensitivity) for T18 – 98% (49/50) , specificity - 100% (297/297).

Targeted Massively Parallel Sequencing (t-MPS)DANSE assay, FORTE algorithm

Case control study involving 3228 pregnancies. Detection rate (Sensitivity) for T21 – 100% (81/81), false-positive rate 0.03% (1/2888).

4.6% test failure.

Detection rate (Sensitivity) for T18 – 97% (37/38) , false-positive rate 0.07% (2/2888).

Targeted Massively Parallel Sequencing (t-MPS)DANSE assay, FORTE algorithm

Targeted Massively Parallel Sequencing (t-MPS)DANSE assay, FORTE algorithm, Microarray

691 were disomic ，18 were trisomy 13 ，37 were trisomy 18 ，132 were trisomy 21.

Targeted Massively Parallel Sequencing (t-MPS)DANSE assay, FORTE algorithm, Microarray

Targeted Massively Parallel Sequencing (t-MPS)DANSE assay, FORTE algorithm, Microarray

parental genotypes

- Parental SupportTM

crossover frequency data (Hapmap Database)

billions of possible monosomic, disomic, and trisomic fetal genotypes at measured loci

fetal cfDNA fraction

expected sequencing data

bayesian statistics are used to determine the relative likelihood of each hypothesis

observed Sequencing data

hypothesis with the maximum likelihood is selected as the copy number and fetal fraction,absolute likelihood of the call is the calculated accuracy, analogous to a test-specific risk score.

multiplex PCR and NGS

- More than 11,000 SNPs covering chr 13,18,21,X,Y are amplified by multi-plex PCR and sequenced by NGS

Read depth

Amplication bias

Noise parameters

Single nucleotide polymorphism (SNP)-based approaches

- Sensitivity and Specificity (145/166) - 100%- 11/11 Trisomy 21- 3/3 Trisomy 18- 2/2 Trisomy 13- 1/1 45,X- 2/2 47,XXY

- Mean calculated accuracy - 99.9%- 21/166 (12.7%) samples failed DNA quality test

Single nucleotide polymorphism (SNP)-based approaches

- 25/25 Trisomy 21- 3/3 Trisomy 18- 1/1 Trisomy 13- 2/2 45,X- 1/1 Triploidy

- Sensitivity and Specificity (229/242) - 100%

- Mean calculated accuracy - 99.9%- 13/242 (5.37%) samples failed DNA quality test

Single nucleotide polymorphism (SNP)-based approaches

A binary hypothesis t-test and logarithmic likelihood ratio L-score

Detection Rate False Positive Rate

T21

T18

100% (139/139)

100% (41/41)

0.03% (1/2820)

0.03% (1/2820)

Shotgun Massively Parallel Sequencing (s-MPS)

Z-score, Z-score with GC correction, Z-score with internal reference chromosome

Shotgun Massively Parallel Sequencing (s-MPS)

Z-score with GC correction

Shotgun Massively Parallel Sequencing (s-MPS)

Z-score with GC correction

Shotgun Massively Parallel Sequencing (s-MPS)

Models for Incorporation of NIPT into Clinical Practice

False positive decrease

False negative rate wont change

‘atypical’ chromosomal abnormalities will be missed

10 weeks

12 weeksImproved overall sensitivity Reduced invasive

testing rate

false negative false positive

expensive

Models for Incorporation of NIPT into Clinical Practice

Increase detection rate to some extent

Reduce invasive testing rate to some extent

Models for Incorporation of NIPT into Clinical Practice

End

NIPT (Non-Invasive Prenatal Testing)Digital PCR