detection of low-frequent mitochondrial dna variants using … · 2019. 4. 10. · detection of...

Detection of low-frequent mitochondrial DNA variants using SMRT sequencing

Marjolein J.A. Weerts

SMRT Leiden 2018 June 13

Content

Mitochondrial DNA & liquid biopsy in oncology

Pitfalls when studying human mitochondrial DNA

Approach to detect low-frequent mitochondrial DNA variants using SMRT sequencing

Application in a pilot study

2

Human mitochondrial DNA

1 * With the exception of aneuploid tumor cells and germ cells

Nuclear DNA

Mitochondrial DNA

Inheritance Maternal & Paternal Maternal

Structure Double stranded lineair

Double stranded circular

Size 3.1 Gb 16.6 kb

Cellular content 99 – 99.9% 1 – 0.1%

Copies/cell Two* Hundreds to Thousands

Gene density ~ 1 / 120 kb ~ 1 / 0.45 kb

Mutation rate ~10-fold higher

Liquid biopsy in oncology

Less invasive than a regular biopsy! Blood-circulating cell-free DNA (cfDNA) Released by normal as well as tumor cells

Genetic information on the patient’s cancer (primary tumor ánd metastases)

JCO 32(6): 579-586, 2014

3

Studying human mtDNA in cancer

HYPOTHESIS

The high mtDNA copy number per cell as well as the high mtDNA mutation rate make it worthwhile to explore the potential of tumor-specific cf-mtDNA variants as cancer marker in the blood of cancer patients

4


HYPOTHESIS


AIMS

Detection of low-frequent mtDNA variants

Highly mtDNA-specific detection of mtDNA variants

4

Pitfalls when studying human mtDNA

High mutation rate

Heteroplasmy

Nuclear insertions of mitochondrial origin: NUMTs

5


High mutation rate Several orders of magnitude higher than that of nDNA

Nearly 10,000 variable positions reported in databases (e.g. mitomap, mtDB)

6


Heteroplasmy Genetically different mtDNA molecules within a single cell

Heteroplasmy patterns differ between tissues within an individual

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Nuclear insertions of mitochondrial origin: NUMTs Each 175 bp mtDNA segment: ~9.5 NUMT copies

Cancer cells contain somatic NUMT insertion events

NUMT similarity to mtDNA interferes with variant detection

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HYPOTHESIS


AIMS



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Minimize the interference of NUMTs:

pure mtDNA sample (devoid of nuclear DNA)

sequence large fragments SMRT sequencing (~80% of human NUMTs are < 500 bp mtDNA segments)

11 MJA Weerts et al., Scientific Reports 2018; 8:2261

SMRT sequencing approach for mtDNA

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1 • Amplification of mtDNA (15-18 PCR cycles)

• 9x 1700-3000 bp amplicons covering the entire mtDNA

2 • Purify & pool amplicons (equimolar) per sample

3 • Barcode each sample (5 PCR cycles), purify & pool samples

4 • Generate SMRTbell library

5 • Sequencing on RSII or Sequel

MJA Weerts et al., Scientific Reports 2018; 8:2261


13

6 • Generate circular consensus reads (CCS2 algorithm)

7 • Attribute reads using sample-specific barcode (TSSV)

8 • Select highly accurate CCS reads (QV>=99%, >= 5 passes)

9 • Trim reads (Cutadapt) and align to extended version or rCRS

(BWA-MEM)

10 • Call positions alternative to the reference sequence in pileup

(Rsamtools, min_nucleotide_depth=5)



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(BWA-MEM)




compensate for mapping bias due to

circularity of the mitochondrial

genome


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(BWA-MEM)






Determine detection limits empirically:

mixtures of the cell lines containing different mtDNA variants



Determine detection limits empirically:

mixtures of the cell lines containing different mtDNA variants

Allele frequency

0% 0.001% 0.01% 0.1% 1% 10%

Digital PCR 0/2 0/2 1/2 1/2 2/2 2/2

UltraSEEK 0/7 0/7 0/7 2/7 7/7 7/7

SMRT seq 0/23 0/23 0/23 21/23 23/23 23/23 positions


HYPOTHESIS


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Tumor-specific cf-mtDNA variants as cancer marker in the blood of cancer patients?

15 MJA Weerts et al., Neoplasia. 2018; 20(7):687–696

Pilot study: SMRT sequence the entire mtDNA of eight cancer patients

Tumor, matched normal (tissue of origin!) and cfDNA


Patient 1

Primary breast cancer (2 cm) with lymph nodes involved

Neoadjuvant endocrine therapy

Surgery, radiation

Prolonged endocrine therapy

Disease recurrence after 4 months (bone, lung and liver metastases)

Second-line endocrine therapy

Disease progression

Chemotherapy



Patient 1



Surgery, radiation




Disease progression

Chemotherapy

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Normal mammary Primary tumor

cfDNA

cfDNA

MJA Weerts et al., Neoplasia. 2018; 20(7):687–696

1200

G>A

5351

A>G

7368

T>C

664

G>A

6255

G>A

1310

3 G

>A

1629

8 T>

C

Primary tumor a 0 0 0 15% 44% 1.2% 99%

Primary tumor b 0 0 0 7.6% 38% 1.2% 99%

Mammary tissue a 0 0 0 0 0.8% 0 99%

Mammary tissue b 0 0 0 0 0 0 98%

cfDNA (pre surgery) 2.0% 1.1% 1.0% 0 0 0 99%

cfDNA (post chemo) 2.9% 0 0 0 0 0 99%


Patient 1



Patient 1



Surgery, radiation




Disease progression

Chemotherapy

18

Normal mammary Primary tumor

cfDNA

cfDNA

cfDNA

cfDNA cfDNA


1200

G>A

5351

A>G

7368

T>C

664

G>A

6255

G>A

1310

3 G

>A

1629

8 T>

C

Primary tumor a 0 0 0 15% 44% 1.2% 99%

Primary tumor b 0 0 0 7.6% 38% 1.2% 99%

Mammary tissue a 0 0 0 0 0.8% 0 99%

Mammary tissue b 0 0 0 0 0 0 98%

cfDNA (pre surgery) 2.0% 1.1% 1.0% 0 0 0 99%

cfDNA (pre 2nd line) na na na 0 0.03% na na

cfDNA (pre chemo) na na na 0.06% 0.3% na na

cfDNA (post chemo) na na na 0 0 na na

cfDNA (post chemo) 2.9% 0 0 0 0 0 99%


Patient 1



Patient 2

Colorectal cancer with synchronous hepatic metastases

Surgery



Patient 2

Colorectal cancer with synchronous hepatic metastases

Surgery

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Normal colon Primary tumor cfDNA

Metastasis 1

Metastasis 2 Normal liver


7078

G>A

1245

3 T>

C

1016

0 C

>T

1091

4 G

>A

66 G

>T

152

T>C

189

A>G

1614

7 C

>T

1614

8 C

>T

1924

T>C

2305

T>C

4048

G>A

1630

4 T>

C

60 T

>C

72 T

>C

Primary tumor 0 0 0 0 0 0 0 0 0 0.7%

9.9%

1.4%

99%

0 11%

Metastasis 1 0 0 0 11%

0 0 0 0 0 42%

34%

0 100%

0 6.2%

Metastasis 2 0 0 21%

1.4%

0 0 0 0 0.9%

35%

39%

0 100%

0.7%

5.6%

Colon tissue 0 0 0 0 2.5%

1.1%

0.4%

0 0 0 1.1%

0 99%

0 6.5%

Liver tissue 0 0 0 0 0 0.4%

1.1%

1.5%

1.2%

0 0 0 99%

3.4%

43%

cfDNA 1.2%

1.9%

0 0 0 0 0.2%

0 0 0 0 0 99%

0 0.2%


Patient 2



HYPOTHESIS


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HYPOTHESIS


CONCLUSIONS

SMRT sequencing suitable for low-frequent mtDNA single-nucleotide variant detection.

Tumor-specific mtDNA variants rarely detected as cfDNA.

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Acknowledgements

These results have been published

Weerts et al., Scientific Reports 2018; 8:2261 Weerts et al., Neoplasia. 2018; 20(7):687–696

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Erasmus MC Medical Oncology John Foekens

John Martens

Stefan Sleijfer

LUMC Leiden Genome Technology Center Rolf Vossen

Yahya Anvar

Philips Research Precision and

Decentralized Diagnostics Dianne van Strijp

Pieter-Jan van der Zaag

Eveline den Biezen – Timmermans

Anja van de Stolpe

contact: [email protected]

detection of low-frequent mitochondrial dna variants using … · 2019. 4. 10. · detection of...

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