IJC
`
`International Journal of Cancer
`
`Establishment of tumor-specific copy number alterations from
`plasma DNA of patients with cancer
`
`Ellen Heitzer1*, Martina Auer1*, Eva Maria Hoffmann1*, Martin Pichler2*, Christin Gasch3, Peter Ulz1, Sigurd Lax4,
`Julie Waldispuehl-Geigl1, Oliver Mauermann3, Sumitra Mohan1, Gunda Pristauz5, Carolin Lackner6, Gerald H€ofler6,
`Florian Eisner2, Edgar Petru5, Heinz Sill7, Hellmut Samonigg2, Klaus Pantel3, Sabine Riethdorf3, Thomas Bauernhofer2,
`Jochen B. Geigl1 and Michael R. Speicher1
`
`1 Institute of Human Genetics, Medical University of Graz, Harrachgasse 21/8, A-8010 Graz, Austria
`2 Division of Oncology, Medical University of Graz, Auenbruggerplatz 15, A-8036 Graz, Austria
`3 Institute of Tumor Biology, University Medical Center Hamburg Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany
`4 Department of Pathology, General Hospital Graz West, Goestingerstrasse 22, A-8020 Graz, Austria
`5 Department of Obstetrics and Gynecology, Medical University of Graz, Auenbruggerplatz 14, A-8036 Graz, Austria
`6 Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, A-8036 Graz, Austria
`7 Division of Hematology, Medical University of Graz, Auenbruggerplatz 38, A-8036 Graz, Austria
`
`With the increasing number of available predictive biomarkers, clinical management of cancer is becoming increasingly reliant on
`the accurate serial monitoring of tumor genotypes. We tested whether tumor-specific copy number changes can be inferred from the
`peripheral blood of patients with cancer. To this end, we determined the plasma DNA size distribution and the fraction of mutated
`plasma DNA fragments with deep sequencing and an ultrasensitive mutation-detection method, i.e., the Beads, Emulsion, Amplifica-
`tion, and Magnetics (BEAMing) assay. When analyzing the plasma DNA of 32 patients with Stage IV colorectal carcinoma, we found
`that a subset of the patients (34.4%) had a biphasic size distribution of plasma DNA fragments that was associated with increased
`circulating tumor cell numbers and elevated concentration of mutated plasma DNA fragments. In these cases, we were able to estab-
`lish genome-wide tumor-specific copy number alterations directly from plasma DNA. Thus, we could analyze the current copy num-
`ber status of the tumor genome, which was in some cases many years after diagnosis of the primary tumor. An unexpected finding
`was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quanti-
`ties. When we analyzed plasma DNA from 35 patients with metastatic breast cancer, we made similar observations suggesting that
`our approach may be applicable to a variety of tumor entities. This is the first description of such a biphasic distribution in a surpris-
`ingly high proportion of cancer patients which may have important implications for tumor diagnosis and monitoring.
`
`CancerGenetics
`
`the molecular
`Recent advances in the understanding of
`mechanisms of cancer have highlighted the need for person-
`alized medicine approaches not only in terms of prognosis
`but also for diagnostic strategies. Extensive work has resulted
`in the identification of biomarkers that have been imple-
`mented in cancer clinical practice at several levels: prognos-
`tics,
`predictive
`and
`pharmacokinetic
`biomarkers.
`For
`
`example, in colorectal cancer (CRC), the KRAS mutations in
`Exon 2 (Codons 12 and 13) represent a paradigm that has
`been established as a negative predictive marker for treatment
`with epidermal growth factor receptor (EGFR) inhibitors
`such as cetuximab and panitumumab.1 The discovery of fur-
`ther biomarkers will be accelerated by the recent advances in
`cancer genomics.
`
`Key words: plasma DNA, tumor monitoring, predictive and prognostic biomarker, copy number changes
`Additional Supporting Information may be found in the online version of this article.
`*E.H., M.A., E.M.H. and M.P. contributed equally to this work
`Thomas Bauernhofer’s current address is: LKH Leoben, Department for Hematology and Oncology, Vordernberger Straße 42, A-8700 Leo-
`ben, Austria
`Grant sponsor: European Commission (GENINCA); Grant number: 202230; Grant sponsor: Austrian Science Fund (FWF); Grant
`numbers: P20338, W 1226-B18 (DKplus Metabolic and Cardiovascular Disease); Grant sponsor: COMET Center ONCOTYROL; Grant
`sponsor: Oesterreichische Nationalbank, Anniversary Fund; Grant number: 14066; Grant sponsor: European Research Council Advanced
`Investigator Grant DISSECT
`DOI: 10.1002/ijc.28030
`History: Received 23 Oct 2012; Accepted 17 Dec 2012; Online 15 Jan 2013
`Correspondence to: Jochen B. Geigl, Institute of Human Genetics, Medical University of Graz, Harrachgasse 21/8, A-8010 Graz, Austria,
`Tel.: 143-316-380-4110, Fax: 143-316-380-9605, E-mail: jochen.geigl@medunigraz.at or Michael R. Speicher, Institute of Human Genetics,
`Medical University of Graz, Harrachgasse 21/8, A-8010 Graz, Austria, Tel.: 143-316-380-4110, Fax: 143-316-380-9605,
`E-mail: michael.speicher@medunigraz.at
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`What’s new?
`Tumors shed their DNA into the bloodstream. This DNA can be detected, but whether it’s useful as a diagnostic tool hasn’t
`been clear from existing reports. Rather than attempt to pick out specific mutations, however, this study asked whether it
`would be possible to get a genome-wide view of tumor-specific copy number changes from this circulating tumor cell DNA.
`When they analyzed the plasma DNA of patients with colorectal cancer, the authors found that about a third of the patients
`had plasma DNA that fell into two distinct size categories, and this correlated with higher numbers of circulating tumor cells.
`They could then detect tumor-specific copy-number changes from this plasma DNA. Further developing this non-invasive acqui-
`sition of tumor material could aid in tailoring specific disease treatment strategies.
`
`Access to accurate and sensitive methods for the detection of
`such predictive biomarkers is of utmost importance to the clini-
`cal oncologist. Numerous studies have tried to identify such bio-
`markers in the form of circulating DNA because tumors shed
`DNA into the circulation that can be detected by appropriate
`means. However, early reports proposing that the presence or
`absence of circulating DNA or its concentration was of diagnos-
`tic value2,3 have been called into question.4–7 Analyses of
`plasma/serum DNA for loss of heterozygosity8,9 or for tumor-
`related methylation patterns10,11 often lack specificity. Further-
`more, it has been proposed that the detection of mutations in
`peripheral blood, which have previously been identified in the
`corresponding primary tumor from the same patient, may pro-
`vide a specific biomarker of disease burden. Therefore, multiple
`studies focused on the detection of such specific and predeter-
`mined mutations.12–18 For example, the emergence of KRAS
`mutant clones as evidence for acquired resistance to targeted
`EGFR blockade in patients with CRC has been inferred from
`the analysis of plasma DNA.19,20 A broader approach is the use
`of targeted amplicon sequencing for the simultaneous analysis
`of multiple cancer driver genes.21 However, the sensitive and
`specific detection of a mutated base in a vast excess of normal
`DNA requires specialized techniques that are currently beyond
`the scope of many diagnostic laboratories.12–14,18
`In our study, we investigated whether instead of a targeted
`approach a genome-wide view of tumor-specific copy number
`changes can be established from peripheral blood, i.e., plasma
`DNA from patients with cancer. To this end, we simultaneously
`quantified the normal and mutant DNA molecules in a given
`sample and established genome-wide copy number changes
`from plasma and compiled detailed data on copy number
`changes in relation to the respective primary tumors and metas-
`tases. We validated these observations with blood samples from
`35 patients with breast cancer. Our results suggest that complex
`tumor genomes can be reconstructed from the peripheral blood
`of patients with cancer. Our approach may be of interest for two
`scenarios: first, as a research tool to investigate a tumor genome
`at late stage disease, which is not commonly studied. Second, it
`may pave the way for new disease monitoring strategies.
`
`Material and Methods
`Preparation of plasma DNA
`Whole blood (9 ml) was collected in routine ethylenediamine
`tetraacetic acid (EDTA) Vacutainer tubes (BD Biosciences,
`Heidelberg, Germany). To stabilize cell membranes and to
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`impede cell lysis, 0.225 ml of a 10% neutral buffered solution
`containing formaldehyde (4% weight per volume) (Sigma-
`Aldrich, Vienna, Austria) was added immediately after blood
`withdrawal. Blood samples were gently inverted, stored at
`room temperature and further processed within 2 hr. Plasma
`was prepared according to Ref. 22. In brief, tubes were cen-
`trifuged at 200g for 10 min with the brake and acceleration
`powers set to zero, followed by a subsequent centrifugation
`step at 1600g for 10 min. The supernatant was collected,
`transferred to a new 15 ml tube and spun at 1600g for 10
`min. The plasma was carefully aliquoted into new 2 ml
`Eppendorf tubes and stored at 280C.
`DNA was
`isolated from plasma samples using the
`QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) or
`the Qiagen Circulating Nucleic Acids Kit (CNA) (Qiagen,
`Hilden, Germany) according to the manufacturer’s instruc-
`tions. DNA was eluted in 30 ll of distilled water for Mini
`Kit extractions and in 100 ml for CNA Kit, respectively.
`
`Further methods
`All other methods used in this article are detailed in the Sup-
`porting Information.
`
`Results
`A subset of patients with CRC had a biphasic
`plasma DNA size distribution
`We analyzed the peripheral blood of 32 patients with advanced
`stage CRC (Supporting Information and Supporting Information
`Table 1). We started with measuring of the plasma DNA concen-
`trations of healthy controls and patients with advanced-stage
`CRC. Compared to controls (mean: 15.21 ng/ml; median: 14.37
`ng/ml; range: 12.20–19.51 ng/ml), patients showed invariably
`higher values with substantial variability (mean: 275.35 ng/ml;
`median: 139.0 ng/ml; range: 22.44–1,037.49 ng/ml) (p<0.0001).
`Because mutant DNA fragments in the blood circulation of
`cancer patients were reported to be degraded relative to non-
`mutant DNA fragments,12 we used a microfluidics-based plat-
`form for sizing. We observed an enrichment of plasma DNA
`fragments within the range of 85–230 bp in the healthy con-
`trols (Fig. 1a, upper plot). Plasma DNA fragments within this
`size range have been associated with the release of DNA from
`apoptotic cells after enzymatic processing.12,23 There was no
`significant qualitative difference regarding the size distribution
`of plasma DNA fragments between the healthy controls and
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`Figure 1. Characteristics of plasma DNA from healthy controls and patients with advanced-stage colorectal cancer (CRC). (a) Size distribu-
`tion of plasma DNA fragments from a healthy donor (upper plot), Patient #11 (center plot) and Patient #22 (lower plot). Normalization was
`performed using two internal markers, visible as high, narrow amplitudes at Positions 35 and 10.380 bp, respectively. For each analysis,
`800 pg of DNA was used. (b) Patients with a biphasic plasma DNA size distribution (with bi. pDNA) have higher plasma DNA concentrations
`compared to patients lacking the second peak (without bi. pDNA). (c) The occurrence and number of CTCs is closely correlated with a
`biphasic plasma DNA size distribution. (d) Deep sequencing using three different sequencing reaction sizes (i.e., 119, 168 and 323 bp)
`identified few mutated KRAS fragments in patients without a biphasic plasma DNA size distribution (blue), but high levels of mutated KRAS
`fragments in patients with a biphasic plasma DNA size distribution (red) (the errors bars represent SDs).
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`21 (65.6%) of the CRC cases despite the higher concentrations
`of plasma DNA in the latter group (Fig. 1a, center plot). How-
`ever, in 11 (34.4%) patients, we observed a second peak con-
`sisting of DNA fragments with a size range of 240 to 400 bp,
`or even longer in some cases (Fig. 1a, lower plot). Interest-
`ingly, the patients with biphasic plasma DNA size distributions
`had significantly higher plasma DNA concentrations (mean:
`604 ng/ml; median: 562 ng/ml; range: 260–1,037 ng/ml) than
`patients without a second peak (mean: 103 ng/ml; median: 89
`ng/ml; range: 22–201 ng/ml) (p<0.0001; Fig. 1b).
`
`The biphasic plasma DNA size distribution correlates with
`circulating tumor cell occurrence
`To investigate whether the DNA size distributions detected
`might reflect the contributions of different tumor cell popula-
`tions releasing their DNA into the circulation around the
`time of blood collection, we determined the number of circu-
`lating tumor cells (CTCs). We used the FDA-approved Veri-
`dex system for CTC detection24 in 30 of the 32 patients (the
`analysis could not be performed for patients #10 and #35).
`We observed a clear correlation between the presence of a
`biphasic plasma DNA size distribution and CTC occurrence.
`In the patient group with a biphasic distribution (n510), we
`found a mean number of 52 CTCs (median: 35; range: 0–
`181). In contrast, the mean CTC number in patients without
`a biphasic plasma DNA size distribution (n520) was only
`1.5 (median: 1; range: 0–7) (p50.0003; Fig. 1c).
`
`Deep sequencing of plasma DNA for KRAS mutations
`To investigate whether mutant DNA fragments were present in
`both the first and second peaks in the plasma DNA fragment size
`distribution, we used ultra-deep pyrosequencing. We used eight
`patient samples with and without biphasic plasma DNA size dis-
`tributions and KRAS mutations in their corresponding primary
`tumors to establish the percentage of mutated plasma DNA frag-
`ments for different sequencing reaction sizes (i.e., 119, 168 and
`323 bp). In four patients (#6, #10, #25 and #38) with biphasic
`plasma DNA size distributions, we found a high percentage of
`mutated DNA fragments compared to the other four patients
`without biphasic peak (#7, #11, #15 and #16) (Fig. 1d; Supporting
`Information Table 2). With the exception of Patient #25, the frac-
`tion of mutant molecules was not dependent on the size of the
`amplicon, suggesting that for these patients (#6, #10 and #38)
`DNA from the first and the second peaks consists largely of tu-
`mor DNA. By contrast, in the four patients who had only the first
`apoptosis-related peak deep sequencing identified mutated KRAS
`fragments at low levels in two cases (#11 and #15) and none in
`the other two patients (#7 and #16) (Fig. 1d; Supporting Informa-
`tion Table 2). In these four patients, mutated DNA fragments
`were not observed in the 323 bp sequencing reaction.
`
`Genome-wide estimation of copy number
`changes in plasma DNA
`We postulated that, in addition to KRAS-mutated DNA frag-
`ments, other tumor DNA fragments should be present in the
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`circulation of these patients and that these might provide
`insights about the tumor genome. To address this hypothesis,
`we generated random DNA libraries by converting the plasma
`DNA fragments into polymerase chain reaction (PCR)-ampli-
`fiable OmniPlex Library molecules flanked by universal pri-
`ming sites
`for whole-genome amplification (WGA) and
`subjected the WGA products to array CGH on a 60 K micro-
`array platform. This array platform consists of 55,077 oligo-
`nucleotides,
`and we
`calculated
`for
`each
`of
`these
`oligonucleotides whether the ratio values were decreased, bal-
`anced or increased. Plasma DNA from six healthy controls
`showed a mean of 4,026 oligonucleotides from the 55,077 oli-
`gonucleotides (7.2%; range: 3,606–4,321; 6.4%–7.7%), with
`aberrant ratio values for the autosomes (Fig. 2a) (details about
`these calculations are in the Supporting Information). When
`we performed array CGH with plasma DNA from patients
`who had only the first plasma DNA peak (n521), we
`observed an increase in oligonucleotides with aberrant ratio
`values with a mean of 5,572 oligonucleotides (10.0%; range:
`4,587–7,469; 8.2%–13.4%; p50.001 compared to the healthy
`controls) (Fig. 2b). Because these oligonucleotides involved
`only single, nonadjacent oligonucleotides, they most likely
`represented artifacts of the fragmentation and/or amplifica-
`tion process. In contrast, the plasma DNA in 10 of the 11
`patients with a second peak had a mean number of oligonu-
`cleotides with copy number changes of 12,476 (22.3%; range:
`9,117–14,915; 16.3%–26.7%), which differed highly signifi-
`cantly from both the aforementioned CRC cases and the
`healthy controls (p<0.0001 each) (Fig. 2c).
`
`Tumor-specific, genome-wide imbalances in plasma DNA
`with a biphasic size distribution
`Because most of the aforementioned copy number changes in
`the plasma DNA with biphasic size distribution involved
`large regions of adjacent oligonucleotides, we determined
`whether or not they were tumor specific by comparing them
`to copy number changes of the primary tumor and, if avail-
`able, metastatic material. As an exemplary case, we present
`Patient #6 in greater detail. When the initial diagnosis was
`made in Patient #6, he already had metastases in the liver,
`bones, abdominal lymph nodes and peritoneum. At this time,
`only a biopsy was obtained from the primary tumor. Five
`months later, a cerebellar metastasis was completely resected,
`and 3 months after that, we collected blood for our analy-
`ses—i.e., 8 months after the initial diagnosis. Thus, multiple
`tumor sites could have released tumor DNA into the circula-
`tion at the time of blood collection.
`We noted marked copy number differences between the
`primary tumor and the cerebellar metastasis in Patient #6
`(Supporting Information Fig. 1, Panels 1 and 2), indicative of
`the presence of several malignant cell clones. We found
`numerous copy number changes in the plasma DNA (Sup-
`porting Information Fig. 1, Panel 3) and performed a detailed
`analysis consisting of the following steps: First, we compared
`the ratio of the profiles of the available material, i.e., primary
`colorectal tumor, cerebellar metastasis and plasma DNA (Fig.
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`Figure 2. Heat maps of plasma DNA profiles from healthy controls and patients with advanced-stage colorectal cancer (CRC). (a) Heat map
`of plasma DNA profiles from healthy donors (black: balanced; red: under-represented; green: over-represented). As we used male reference
`DNA in all experiments, female plasma DNA samples have a relative over-representation of the X chromosome and an under-representation
`of the Y-chromosome (F1–F3), whereas male samples have balanced sex chromosomes (M1–M3). (b) Heat maps of six exemplary patients
`(#7, #11, #14, #24, #28 and # 37) that lack a biphasic plasma DNA size distribution. (c) Heat maps of plasma DNA from 10 patients (#6,
`#9, #10, #18, #20, #22, #25, #26, #27 and #33) that have a biphasic plasma DNA size distribution.
`
`showing
`3a). We then constructed detailed heat maps
`whether the copy number status was decreased, balanced or
`increased for each oligonucleotide on our array platform (Fig.
`3b). Finally, we determined whether the copy number status
`for each oligonucleotide occurred only in the plasma DNA or
`also in the primary tumor and/or metastasis (Fig. 3c). These
`analyses revealed that the copy number status of 46.3% of
`the oligonucleotides on our array platform was present in all
`three lesions; 18.1% was shared by the primary tumor and
`plasma DNA, 18.0% was shared by the metastasis and plasma
`DNA and 17.6% was unique to the plasma DNA (Fig. 3c).
`This suggested that the observed changes in the plasma DNA
`were tumor-specific and were largely caused by clones both
`from the primary and the cerebellar metastatic tumor. As the
`cerebellar metastasis had been removed, cells from this meta-
`static clone are apparently still present
`in the patient.
`Whether the copy number changes observed only in the
`plasma DNA could have resulted from the other metastases
`or from parts of the primary tumor that were not included
`in our analysis remains unknown.
`
`In addition to Patient #6, we had three other patients (i.e.,
`#9: Supporting Information Fig. 2; #26: Supporting Information
`Fig. 3; #33: Supporting Information Fig. 4) for whom material
`from the primary tumor and metastases were available for com-
`parison. Altogether, the plasma DNA samples from these four
`patients (i.e., #6, #9, #26 and #33) displayed an identical copy
`number status (i.e., lost, balanced and gained) in all three sam-
`ples (primary, metastasis and plasma DNA) for an average of
`54.7% (median: 54.3%; range: 46.3%–63.6%) of all oligonucleo-
`tides. About 10% (median: 9.4%; range: 3.1%–18.1%) of plasma
`DNA copy number changes were unique to the metastasis and,
`vice versa, 14.5% (median: 16.2%; range: 2.9%–22.7%) were
`only present in the primary tumor. About 20.9% (median:
`18.7%; range: 15.7%–30.4%) of plasma DNA changes were
`observed only in the plasma DNA but not in the primary tumor
`and metastasis. However, all four patients had additional me-
`tastases at various sites (Supporting Information Table 1),
`which were not accessible to us. Thus, copy number changes
`observed only in the plasma DNA could reflect alterations from
`a metastatic site not included in our analysis.
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`Figure 3. Comparison of copy number changes in the primary tumor, metastasis and plasma DNA of Patient #6. (a) Comparison of the ratio
`profiles of representative chromosomes (1, 3, 4, 8, 10, 16, 18 and 20; indicated by numbers below the copy number changes) between
`the primary tumor (PT, left column), metastasis (M, center column) and plasma DNA (Pl, right column). The single green and red bars sum-
`marize the regions that were gained or lost based on all iterative calculations of our algorithm (Supporting Information). The black profile
`regions represent balanced regions, lost regions appear in red and gained regions are shown as green (complete profiles are depicted in
`Supporting Information Fig. 1a). (b) Heat maps comparing the copy number changes in the primary tumor (PT), metastasis (M), and plasma
`DNA (Pl; Black: balanced regions; red: under-represented regions; green: over-represented regions). (c) The bar chart combines information
`on copy number changes and their occurrence in the primary tumor, metastasis and plasma DNA. It displays the percentages of chromo-
`somal regions that were commonly lost (red), balanced (black) or gained (green) in all three samples, shared by metastasis and plasma
`DNA only (blue), shared by primary tumor and plasma DNA only (yellow) or unique to the plasma DNA (gray).
`
`For another patient (#27), we had material from the pri-
`mary tumor only, and we obtained comparable results with
`the plasma DNA, i.e., copy number changes similar to those
`observed in the primary tumor (Supporting Information Fig.
`5). By contrast, we did not find copy number changes of
`large contiguous chromosomal regions in the plasma DNA of
`Patient #38 that unequivocally correspond to those of the pri-
`mary tumor or metastases (Supporting Information Fig. 6).
`
`Plasma DNA analysis in the absence of material from the
`primary tumor or metastasis
`In three cases, i.e., #10 (Supporting Information Fig. 7a), #20
`(Supporting Information Fig. 7b) and #25 (Supporting Infor-
`mation Fig. 7c), no further tumor material was available for
`analyses. However, in each of these cases, we were able to es-
`tablish array-CGH profiles
`reminiscent of copy number
`changes frequently observed in colon cancer (http://www.pro-
`genetix.net), such as loss on 8p and gains on 8q and 20.
`In two cases (i.e., #18, #22),
`insufficient material was
`available because only small biopsies had been taken at the
`time of diagnosis. In Patient #18, our plasma DNA analysis
`again revealed typical CRC-related copy number changes,
`such as loss on 8p and gains on 8q and 20 (Fig. 4a), whereas
`
`in Patient #22, our plasma DNA analysis revealed losses on
`chromosomes 3, 4, 5, 8p and 18 and gains on chromosomes
`7p, 17q and 20 (Fig. 4b). These results suggest that tumor-
`specific copy number profiles can be established even in cases
`where for various reasons no material from the primary tu-
`mor or metastasis is available.
`
`BEAMing analysis
`As all patients included in our study had metastatic disease
`showing disease progression at the time of blood collection,
`the significant variability of plasma DNA size distribution
`and concentrations as well as CTC number was unexpected.
`Furthermore, we were puzzled that even deep sequencing did
`not find evidence for overt mutant DNA fragments in some
`of our patients. Therefore, we tested patients for the presence
`of KRAS mutations with a more sensitive approach,
`i.e.,
`BEAMing, which has the capacity to detect and enumerate
`mutant and wild-type DNA when present at ratios greater
`than 1:10,00012,25 (www.inostics.com). To exclude any varia-
`tion from sample collection and handling, we performed se-
`rial analyses with blood samples from two patients (L1 and
`L2) collected on five (L1) or four (L2) consecutive days.
`These patients had end-stage, highly metastatic colon cancer
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`Figure 4. Array-CGH from two cases where only small biopsies had been taken at the time of diagnosis, so insufficient material was avail-
`able for further analyses. (a) The plasma DNA from Patient #18 revealed typical CRC-related copy number changes, such as loss on 8p and
`gains on 8q and 20. (b) Plasma DNA ratio profile from Patient #22 demonstrating losses on chromosomes 3, 4, 5, 8p and 18 and gains on
`chromosomes 7p, 17q and 20.
`
`and received only palliative treatment (no chemotherapy or
`radiation therapy), which should have no impact on the tu-
`mor burden. In both patients the KRAS G12V mutation had
`been detected in the respective primary tumors.
`In Patient L1, the respective mutant fractions were close
`to the BEAMing detection limit, given as 0.02, on Days 1,
`2, 4 and 5 or even below the detection limit on Day 3 (Fig.
`5a). Accordingly, we observed invariably balanced array-
`CGH profiles on each day. This suggests that even ultra-
`sensitive methods may be incapable of detecting mutant
`DNA fragments in some patients with highly metastatic
`disease.
`fractions were
`the mutant
`in Patient L2,
`In contrast,
`between 6.644 (Day 2) and 11.181 (Day 3) (Fig. 5a). On Day
`3, there was a biphasic plasma DNA size distribution, and we
`observed in the array-CGH analyses copy number changes
`on chromosomes 3, 4, 18, 19 and 20, which we had previ-
`ously also observed in the primary tumor (Fig. 5b). Although
`the plasma DNA size distribution was monophasic on the
`other days, some of these copy number changes were also
`visible in the array-CGH profiles on these days.
`
`Correlation with clinical parameters
`Although this was a pilot study, we then attempted to corre-
`late our findings on plasma DNA size distribution and CTC
`number with clinical parameters. We did not find any corre-
`
`the established CRC tumor
`lation between the levels of
`markers CEA (Supporting Information Fig. 8a) and CA19-9
`(Supporting Information Fig. 8b) and plasma DNA concen-
`tration or CTC numbers. The percentage of patients with a
`biphasic plasma DNA size distribution was 67%, 44% and
`38% for patients with metastases
`in bone (n56),
`liver
`(n525) and peritoneal carcinomatosis (n58), respectively,
`and only 9% for patients with lung metastasis (n511) (Sup-
`porting Information; Supporting Information Table 3). These
`percentages were almost identical for patients with more than
`six CTCs, i.e., 67%, 36%, 9% and 38%, for patients with me-
`tastasis in bone,
`liver,
`lung and peritoneal carcinomatosis,
`respectively. In fact, only one of the 11 patients with lung
`metastasis (Patient #38) demonstrated both a biphasic plasma
`DNA size distribution and a high CTC number, according to
`the Veridex system.
`
`Analyses of plasma DNA from patients with breast cancer
`To test whether our observations can be extended to other
`tumor entities, we collected blood from 35 patients with
`breast cancer. Indeed, we observed the biphasic plasma DNA
`size distribution in 14 (40%) patients, which again correlated
`with the plasma DNA concentration (Fig. 6a) and number of
`CTCs (Fig. 6b). In these cases, we could again reconstruct tu-
`mor specific copy number changes from the plasma DNA
`(Figs. 6c–6d).
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`Figure 5. Summary of the BEAMing analysis and representative array-CGH profiles from one representative patient. (a) Mutant fraction and
`call (i.e., either mutant or wildtype) as established by BEAMing for two patients. (b) The upper panel illustrates the array-CGH profile of the
`primary tumor, the lower panel the corresponding profile established with plasma DNA from Day 3.
`
`Discussion
`A major goal of cancer medicine is to move from fixed treat-
`ment regimens to therapies tailored to a patient’s individual
`tumor. Following recent breakthroughs in genomics technol-
`ogy, major efforts to identify potentially informative muta-
`tions using next-generation sequencing are under way.26
`However, such detailed analyses are usually performed with
`samples obtained during the initial diagnosis and/or metasta-
`sis.27,28 All these genomic studies have confirmed that tumor
`genomes are complex and highly prone to changes. In our
`study, we addressed whether complex tumor genomes may
`be inferred noninvasively from the peripheral blood of
`patients with cancer.
`
`We observed that the plasma DNA and CTCs varied sig-
`nificantly in our cohort, although all patients included in our
`study had metastatic disease showing disease progression at
`the time of blood collection. Some cancer patients with only
`the first peak have higher plasma DNA levels compared to
`healthy controls but, as confirmed by deep sequencing,
`BEAMing and array-CGH, a very low percentage of mutated
`DNA fragments. This is consistent with necrotic neoplastic
`cells being engulfed by macrophages, which involves the kill-
`ing of neoplastic cells and the surrounding stromal and
`inflammatory cells.12 The released DNA contains multiple
`wild-type DNA sequences, which may explain the increase in
`total, nonmutant circulating DNA.
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`Plasma DNA analysis
`
`Figure 6. Evaluation of blood from 35 patients with breast cancer and representative array-CGH profiles. (a) Breast cancer patients with a
`biphasic plasma DNA size distribution (with bi. pDNA) have increased plasma DNA concentrations (here depicted as log10 ratio) compared
`to patients lacking the second peak (without bi. pDNA). (b) The number of CTCs is also increased in patients with a biphasic plasma DNA
`size distribution. (c) Array CGH profile of the primary tumor of breast cancer Patient #20. (d) Corresponding array-CGH profile obtained with
`plasma DNA from Patient #20, who had a biphasic plasma DNA size distribution.
`
`In contrast, a biphasic plasma DNA size distribution may
`indicate a distinct biological process, because its occurrence is
`associated with very high plasma DNA levels, elevated per-
`centages of mutated DNA fragments in the circulation, and
`an increased number of CTCs. The biphasic plasma DNA
`distribution likely reflects massive cell destruction with direct
`
`shedding of DNA from tumor cells and cellular fragments
`into the bloodstream. As the number of CTCs identified by
`the Veridex system is relatively small, CTCs by themselves
`likely contribute only small amounts of tumor DNA into the
`circulation. Hence, the biphasic plasma DNA size distribution
`is more likely due to various mechanism of release at the site
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`for example, when the tumor has invaded
`of the tumor,
`through blood vessels. The first peak consisting of plasma
`DNA fragments within the range of 85–230 bp could be
`released from apoptotic cells after enzymatic processing. Pre-
`viously plasma DNA fragments within this size range had
`been associated with the release of DNA from apoptotic cells
`after enzymatic processing, because the length of these frag-
`ments corresponds approximately to the DNA wrapped
`around a nucleosome (142 bp) plus a linker fragment (20
`bp).12,23 Accordingly, the DNA fragments from the second
`peak likely represent di- and trinucleoso