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Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease.

Olsson E, Winter C, George A, Chen Y, Howlin J, Tang MH, Dahlgren M, Schulz R, Grabau D, van Westen D, Fernö M, Ingvar C, Rose C, Bendahl PO, Rydén L, Borg Å, Gruvberger-Saal SK, Jernström H, Saal LH - EMBO Mol Med (2015)

Bottom Line: Metastatic breast cancer is usually diagnosed after becoming symptomatic, at which point it is rarely curable.Cell-free circulating tumor DNA (ctDNA) contains tumor-specific chromosomal rearrangements that may be interrogated in blood plasma.Using an approach combining low-coverage whole-genome sequencing of primary tumors and quantification of tumor-specific rearrangements in plasma by droplet digital PCR, we identify for the first time that ctDNA monitoring is highly accurate for postsurgical discrimination between patients with (93%) and without (100%) eventual clinically detected recurrence. ctDNA-based detection preceded clinical detection of metastasis in 86% of patients with an average lead time of 11 months (range 0-37 months), whereas patients with long-term disease-free survival had undetectable ctDNA postoperatively. ctDNA quantity was predictive of poor survival.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology and Pathology, Department of Clinical Sciences, Lund University, Lund, Sweden Lund University Cancer Center, Lund, Sweden.

No MeSH data available.


Related in: MedlinePlus

Identification of chromosomal rearrangements and personalized assay designLow-coverage whole-genome sequencing of the primary tumor was used to enumerate chromosomal rearrangements. Shown are results for patient DF1, with inter- and intra-chromosomal rearrangements plotted as a Circos diagram (Krzywinski et al, 2009). Chromosomes 1–22 and X are ordered in the outer circle. From the outside, concentrically, are plotted the DNA copy number estimations from the whole-genome sequencing data and the chromosome ideograms. The orange intra-chromosomal and blue inter-chromosomal arcs in the center indicate chromosomal rearrangements supported by two or more paired-end reads.Circos diagram for patient EM11. Plots for all patient tumors are shown in Supplementary Fig S1.One example rearrangement from patient EM11, indicated in red in (B), with identification of the exact fusion sequence between chromosomes 8p22 and 11q14.1. Aligned sequencing reads are highlighted in blue when its read pair aligns concordantly on the same chromosome or in light green if its read pair aligns on another chromosome. Within each sequencing read, nucleotide bases with exact match to the reference sequence (shown in the middle with yellow shading) are not printed. Mismatching bases are shown in blue if matching to 11q14.1 and green if matching to 8p22. At the bottom, the personalized dual-labeled probe and primers designed for this validated rearrangement are illustrated. F denotes the fluorescent molecule and Q the two quenching molecules.
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fig02: Identification of chromosomal rearrangements and personalized assay designLow-coverage whole-genome sequencing of the primary tumor was used to enumerate chromosomal rearrangements. Shown are results for patient DF1, with inter- and intra-chromosomal rearrangements plotted as a Circos diagram (Krzywinski et al, 2009). Chromosomes 1–22 and X are ordered in the outer circle. From the outside, concentrically, are plotted the DNA copy number estimations from the whole-genome sequencing data and the chromosome ideograms. The orange intra-chromosomal and blue inter-chromosomal arcs in the center indicate chromosomal rearrangements supported by two or more paired-end reads.Circos diagram for patient EM11. Plots for all patient tumors are shown in Supplementary Fig S1.One example rearrangement from patient EM11, indicated in red in (B), with identification of the exact fusion sequence between chromosomes 8p22 and 11q14.1. Aligned sequencing reads are highlighted in blue when its read pair aligns concordantly on the same chromosome or in light green if its read pair aligns on another chromosome. Within each sequencing read, nucleotide bases with exact match to the reference sequence (shown in the middle with yellow shading) are not printed. Mismatching bases are shown in blue if matching to 11q14.1 and green if matching to 8p22. At the bottom, the personalized dual-labeled probe and primers designed for this validated rearrangement are illustrated. F denotes the fluorescent molecule and Q the two quenching molecules.

Mentions: Twenty patients enrolled in the Breast Cancer and Blood Study (BC Blood, Sweden) (Borgquist et al, 2013), an ongoing prospective study at Lund University since 2002, were included in the present investigation for retrospective analysis of ctDNA (Fig1A). Six patients had long-term disease-free survival (9.2 years median follow-up; termed DF patients), and 14 had eventual diagnosis of clinical metastasis from 1.2 to 5.1 years after primary surgery (termed eventual metastatic [EM] patients) (Table1). For each patient, a sample of the primary tumor, a normal tissue sample, and 3–6 blood plasma samples that were collected during the clinical course were available. First, to identify tumor-associated chromosomal rearrangements that could serve as biomarkers, whole-genome sequencing (WGS) was performed on DNA isolated from 21 primary breast tumors (patient EM6 had bilateral primary breast cancers). On average, 93 million DNA fragments were sequenced per tumor (range 54–160 million), yielding a mean genome sequence coverage of 5.3-fold (range 1.8–12.9) and mean physical coverage of 15.6 (range 9.2–28.2) (Supplementary Table S1). We developed an analysis pipeline incorporating our SplitSeq computational method to identify inter- and intra-chromosomal rearrangements using an approach that scanned for paired sequence reads where the two reads aligned to discordant positions in the human genome, or individual reads in a read pair that contained juxtaposed sequences from two disparate genomic regions. Chromosomal rearrangements supported by two or more sequenced fragments could be detected in all primary tumors, and on average, 92 rearrangements were identified per tumor (range 21–305) (Fig2, Supplementary Fig S1 and Supplementary Tables S1 and S2). There was no significant difference in sequence coverage or frequencies of chromosomal rearrangements detected between EM patients and DF patients (Mann–Whitney test), and the numbers of detected rearrangements for these 21 cases are similar to other studies of primary breast tumors (Stephens et al, 2009; Banerji et al, 2012; Nik-Zainal et al, 2012).


Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease.

Olsson E, Winter C, George A, Chen Y, Howlin J, Tang MH, Dahlgren M, Schulz R, Grabau D, van Westen D, Fernö M, Ingvar C, Rose C, Bendahl PO, Rydén L, Borg Å, Gruvberger-Saal SK, Jernström H, Saal LH - EMBO Mol Med (2015)

Identification of chromosomal rearrangements and personalized assay designLow-coverage whole-genome sequencing of the primary tumor was used to enumerate chromosomal rearrangements. Shown are results for patient DF1, with inter- and intra-chromosomal rearrangements plotted as a Circos diagram (Krzywinski et al, 2009). Chromosomes 1–22 and X are ordered in the outer circle. From the outside, concentrically, are plotted the DNA copy number estimations from the whole-genome sequencing data and the chromosome ideograms. The orange intra-chromosomal and blue inter-chromosomal arcs in the center indicate chromosomal rearrangements supported by two or more paired-end reads.Circos diagram for patient EM11. Plots for all patient tumors are shown in Supplementary Fig S1.One example rearrangement from patient EM11, indicated in red in (B), with identification of the exact fusion sequence between chromosomes 8p22 and 11q14.1. Aligned sequencing reads are highlighted in blue when its read pair aligns concordantly on the same chromosome or in light green if its read pair aligns on another chromosome. Within each sequencing read, nucleotide bases with exact match to the reference sequence (shown in the middle with yellow shading) are not printed. Mismatching bases are shown in blue if matching to 11q14.1 and green if matching to 8p22. At the bottom, the personalized dual-labeled probe and primers designed for this validated rearrangement are illustrated. F denotes the fluorescent molecule and Q the two quenching molecules.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4551342&req=5

fig02: Identification of chromosomal rearrangements and personalized assay designLow-coverage whole-genome sequencing of the primary tumor was used to enumerate chromosomal rearrangements. Shown are results for patient DF1, with inter- and intra-chromosomal rearrangements plotted as a Circos diagram (Krzywinski et al, 2009). Chromosomes 1–22 and X are ordered in the outer circle. From the outside, concentrically, are plotted the DNA copy number estimations from the whole-genome sequencing data and the chromosome ideograms. The orange intra-chromosomal and blue inter-chromosomal arcs in the center indicate chromosomal rearrangements supported by two or more paired-end reads.Circos diagram for patient EM11. Plots for all patient tumors are shown in Supplementary Fig S1.One example rearrangement from patient EM11, indicated in red in (B), with identification of the exact fusion sequence between chromosomes 8p22 and 11q14.1. Aligned sequencing reads are highlighted in blue when its read pair aligns concordantly on the same chromosome or in light green if its read pair aligns on another chromosome. Within each sequencing read, nucleotide bases with exact match to the reference sequence (shown in the middle with yellow shading) are not printed. Mismatching bases are shown in blue if matching to 11q14.1 and green if matching to 8p22. At the bottom, the personalized dual-labeled probe and primers designed for this validated rearrangement are illustrated. F denotes the fluorescent molecule and Q the two quenching molecules.
Mentions: Twenty patients enrolled in the Breast Cancer and Blood Study (BC Blood, Sweden) (Borgquist et al, 2013), an ongoing prospective study at Lund University since 2002, were included in the present investigation for retrospective analysis of ctDNA (Fig1A). Six patients had long-term disease-free survival (9.2 years median follow-up; termed DF patients), and 14 had eventual diagnosis of clinical metastasis from 1.2 to 5.1 years after primary surgery (termed eventual metastatic [EM] patients) (Table1). For each patient, a sample of the primary tumor, a normal tissue sample, and 3–6 blood plasma samples that were collected during the clinical course were available. First, to identify tumor-associated chromosomal rearrangements that could serve as biomarkers, whole-genome sequencing (WGS) was performed on DNA isolated from 21 primary breast tumors (patient EM6 had bilateral primary breast cancers). On average, 93 million DNA fragments were sequenced per tumor (range 54–160 million), yielding a mean genome sequence coverage of 5.3-fold (range 1.8–12.9) and mean physical coverage of 15.6 (range 9.2–28.2) (Supplementary Table S1). We developed an analysis pipeline incorporating our SplitSeq computational method to identify inter- and intra-chromosomal rearrangements using an approach that scanned for paired sequence reads where the two reads aligned to discordant positions in the human genome, or individual reads in a read pair that contained juxtaposed sequences from two disparate genomic regions. Chromosomal rearrangements supported by two or more sequenced fragments could be detected in all primary tumors, and on average, 92 rearrangements were identified per tumor (range 21–305) (Fig2, Supplementary Fig S1 and Supplementary Tables S1 and S2). There was no significant difference in sequence coverage or frequencies of chromosomal rearrangements detected between EM patients and DF patients (Mann–Whitney test), and the numbers of detected rearrangements for these 21 cases are similar to other studies of primary breast tumors (Stephens et al, 2009; Banerji et al, 2012; Nik-Zainal et al, 2012).

Bottom Line: Metastatic breast cancer is usually diagnosed after becoming symptomatic, at which point it is rarely curable.Cell-free circulating tumor DNA (ctDNA) contains tumor-specific chromosomal rearrangements that may be interrogated in blood plasma.Using an approach combining low-coverage whole-genome sequencing of primary tumors and quantification of tumor-specific rearrangements in plasma by droplet digital PCR, we identify for the first time that ctDNA monitoring is highly accurate for postsurgical discrimination between patients with (93%) and without (100%) eventual clinically detected recurrence. ctDNA-based detection preceded clinical detection of metastasis in 86% of patients with an average lead time of 11 months (range 0-37 months), whereas patients with long-term disease-free survival had undetectable ctDNA postoperatively. ctDNA quantity was predictive of poor survival.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology and Pathology, Department of Clinical Sciences, Lund University, Lund, Sweden Lund University Cancer Center, Lund, Sweden.

No MeSH data available.


Related in: MedlinePlus