Detection of low-frequent mitochondrial DNA variants using SMRT sequencing

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 Inheritance Structure Nuclear DNA Maternal & Paternal Double stranded lineair Mitochondrial DNA Maternal 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 * With the exception of aneuploid tumor cells and germ cells 1

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) 3 JCO 32(6): 579-586, 2014

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 tumorspecific cf-mtdna variants as cancer marker in the blood of cancer patients 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

Pitfalls when studying human mtdna 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

Pitfalls when studying human mtdna Heteroplasmy Genetically different mtdna molecules within a single cell Heteroplasmy patterns differ between tissues within an individual 7

Pitfalls when studying human mtdna 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 9

Highly mtdna-specific detection of mtdna variants 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 1 2 3 4 Amplification of mtdna (15-18 PCR cycles) 9x 1700-3000 bp amplicons covering the entire mtdna Purify & pool amplicons (equimolar) per sample Barcode each sample (5 PCR cycles), purify & pool samples Generate SMRTbell library 5 Sequencing on RSII or Sequel 12 MJA Weerts et al., Scientific Reports 2018; 8:2261

SMRT sequencing approach for mtdna 6 7 8 9 10 Generate circular consensus reads (CCS2 algorithm) Attribute reads using sample-specific barcode (TSSV) Select highly accurate CCS reads (QV>=99, >= 5 passes) Trim reads (Cutadapt) and align to extended version or rcrs (BWA-MEM) Call positions alternative to the reference sequence in pileup (Rsamtools, min_nucleotide_depth=5) 13 MJA Weerts et al., Scientific Reports 2018; 8:2261

SMRT sequencing approach for mtdna 6 7 Generate circular consensus reads (CCS2 algorithm) Attribute reads using sample-specific barcode (TSSV) 8 9 10 Select highly accurate CCS reads (QV>=99, >= 5 passes) Trim reads (Cutadapt) and align to extended version or rcrs (BWA-MEM) Call positions alternative to the reference sequence in pileup (Rsamtools, min_nucleotide_depth=5) compensate for mapping bias due to circularity of the mitochondrial genome 13 MJA Weerts et al., Scientific Reports 2018; 8:2261

Detection of low-frequent mtdna variants Determine detection limits empirically: mixtures of the cell lines containing different mtdna variants 14 MJA Weerts et al., Scientific Reports 2018; 8:2261

Detection of low-frequent 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 14 MJA Weerts et al., Scientific Reports 2018; 8:2261

Tumor-specific cf-mtdna variants as cancer marker in the blood of cancer patients? Pilot study: SMRT sequence the entire mtdna of eight cancer patients Tumor, matched normal (tissue of origin!) and cfdna 15 MJA Weerts et al., Neoplasia. 2018; 20(7):687 696

Tumor-specific cf-mtdna variants as cancer marker in the blood of cancer patients? 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 16 MJA Weerts et al., Neoplasia. 2018; 20(7):687 696

Tumor-specific cf-mtdna variants as cancer marker in the blood of cancer patients? 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 cfdna Primary tumor Normal mammary cfdna 16 MJA Weerts et al., Neoplasia. 2018; 20(7):687 696

Tumor-specific cf-mtdna variants as cancer marker in the blood of cancer patients? Patient 1 1200 G>A 5351 A>G 7368 T>C 664 G>A 6255 G>A 13103 G>A 16298 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 17 MJA Weerts et al., Neoplasia. 2018; 20(7):687 696

Tumor-specific cf-mtdna variants as cancer marker in the blood of cancer patients? Patient 1 Primary breast cancer (2 cm) with lymph nodes involved cfdna cfdna 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 cfdna Primary tumor Normal mammary cfdna cfdna 18 MJA Weerts et al., Neoplasia. 2018; 20(7):687 696

Tumor-specific cf-mtdna variants as cancer marker in the blood of cancer patients? Patient 1 1200 G>A 5351 A>G 7368 T>C 664 G>A 6255 G>A 13103 G>A 16298 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 MJA Weerts et al., Neoplasia. 2018; 20(7):687 696

Tumor-specific cf-mtdna variants as cancer marker in the blood of cancer patients? Patient 2 Colorectal cancer with synchronous hepatic metastases Surgery 20 MJA Weerts et al., Neoplasia. 2018; 20(7):687 696

Tumor-specific cf-mtdna variants as cancer marker in the blood of cancer patients? Patient 2 Colorectal cancer with synchronous hepatic metastases cfdna Surgery Primary tumor Normal colon Metastasis 1 Normal liver Metastasis 2 20 MJA Weerts et al., Neoplasia. 2018; 20(7):687 696

Tumor-specific cf-mtdna variants as cancer marker in the blood of cancer patients? Patient 2 7078 G>A 12453 T>C 10160 C>T 10914 G>A 66 G>T 152 T>C 189 A>G 16147 C>T 16148 C>T 1924 T>C 2305 T>C 4048 G>A 16304 T>C 60 T>C 72 T>C Primary tumor 0 0 0 0 0 0 0 0 0 0.7 Metastasis 1 0 0 0 11 Metastasis 2 0 0 21 1.4 Colon tissue 0 0 0 0 2.5 0 0 0 0 0 42 0 0 0 0 0.9 1.1 Liver tissue 0 0 0 0 0 0.4 cfdna 1.2 1.9 0.4 1.1 0 0 0 0 0.2 35 9.9 34 39 0 0 0 1.1 1.5 1.2 1.4 99 0 100 0 100 0 99 0 0 0 99 0 0 0 0 0 99 0 11 0 6.2 0.7 5.6 0 6.5 3.4 43 0 0.2 MJA Weerts et al., Neoplasia. 2018; 20(7):687 696

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 tumorspecific cf-mtdna variants as cancer marker in the blood of cancer patients CONCLUSIONS SMRT sequencing suitable for low-frequent mtdna single-nucleotide variant detection. Tumor-specific mtdna variants rarely detected as cfdna. 22

Acknowledgements Erasmus MC Medical Oncology John Foekens John Martens Stefan Sleijfer LUMC Leiden Genome Technology Center Rolf Vossen Yahya Anvar contact: m.weerts@erasmusmc.nl Philips Research Precision and Decentralized Diagnostics Dianne van Strijp Pieter-Jan van der Zaag Eveline den Biezen Timmermans Anja van de Stolpe These results have been published Weerts et al., Scientific Reports 2018; 8:2261 Weerts et al., Neoplasia. 2018; 20(7):687 696 23