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`
`Review
`
`Diagnostic potential of circulating
`nucleic acids for oncology
`
`Carsten Goess!
`
`Approximately a decade ago, the PCR-based detection of extracellular, tumor-derived
`circulating nucleic acids in the plasma and serum of cancerpatients was introduced as a
`noninvasive tool for cancer detection. Although the test criteria, sensitivity and specificity,
`comparefavorably with conventional diagnostic measures, until now the methodical
`ponderousnessofcirculating nucleic acids in plasma and serum analysis preventedit from
`becoming a clinical routine application. However, with rapid technical improvement
`towards automated high-throughput platforms, it is expected that the next 5 years will see
`circulating nucleic acids in plasma and serum analysis integratedinto theinitial diagnosis
`and follow-up monitoring of cancer patients. The hopeis that the use of circulating nucleic
`acids in plasma and serum as a molecular tumor marker and potential profiling tool will
`finally translate into a longer survival and better quality oflife for cancerpatients.
`
`Expert Rev. Mol. Diagn. 3(4), 431-442 (2003)
`
` © Future DrugsLtd. All rights reserved. ISSN 1473-7159
`
`Noninvasive
`nucleic
`acid-based molecular
`diagnosis of cancer using body fluids has
`emerged rapidly over the last 12 years. The
`first of these reports dealt with the detection of
`urothelial cancer by DNA-based analysis of
`exfoliated bladder tumorcells in voided urine
`(1). In selected cases it has been repeatedly
`demonstrated that molecular detection of
`tumor cells in body secretions can precede
`conventional
`diagnosis of primary and
`recurrent malignancy [2,3]. Consequently,
`molecular
`tumor detection methods have
`been ascribed a great potential to impact on
`future therapeutic management of cancer
`patients [4].
`Beyond excreted body fluids such as urine
`and sputum, blood constitutes an attractive
`reservoir for nucleic acid-based detection of
`cancer [4]. Blood can be drawn in a minimally
`invasive manner (classified thereafter as non-
`invasive) and this enables repeated testing, a
`feature especially useful for longitudinal mon-
`itoring of responses to therapy and during fol-
`low-up of oncologic patients. Blood consists
`of
`two main fractions:
`a cellular and a
`plasma/serum fraction. Nucleic acid-based
`cancer detection analyzing the cellular blood
`fraction for circulating tumorcells has been
`
`extensively described elsewhere[5,6]. The focus
`of this
`review is
`the detection of cancer
`patients by analyzing their plasma/serum frac-
`tion for the existence of circulating extracellu-
`lar nucleic acids
`(both DNA and RNA)
`derived from tumoral sources. Cell-free circu-
`lating nucleic acids in the plasma and serum
`(CNAPS)of cancer patients have beeninvesti-
`gated extensively as evidenced by more than
`200 articles on the subject. A recent review
`has summarized detailed results on colorectal,
`pancreatic, breast,
`lung and prostate cancer,
`and has concluded that detection and quanti-
`fication of viral DNA in virus-associated
`malignancies such as nasopharyngeal carci-
`noma (associated with Epstein-Barr virus
`[EBV]) might be closest
`to broad clinical
`application [7-8].
`This review is structured into four sections:
`first, the medical context for the detection of
`tumor-derived CNAPS;
`second,
`a_
`brief
`description of the methodology used and the
`obstacles observed;
`third, a selection of the
`results obtained with different detection
`methodsin various cancer entities; and finally,
`a 5-year outlook into the future, taking into
`account competing methods for noninvasive
`cancer detection in blood samples.
`
`00001
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`

`Goessl
`
`CNAPS: from plant physiology to oncology
`The existence of circulating, noncellular nucleic acids (both
`RNA and DNA)wasfirst described more than 50 years ago (91.
`These findings remained largely unnoticed by the oncological
`community until in the late 1970s, when elevated amounts of
`DNAwere characterized in the serum of cancer patients [10].
`Successful therapy was associated with a decrease in serum DNA
`quantities, however, benign diseases, especially autoimmunedis-
`orders, had also been found to be associated with increased
`amountsof circulating DNA [11]. As a consequence, the malignant
`versus benign origin of the increased DNA concentrations found
`in the plasma/serum of cancer patients remained unknown.
`A group ofSwiss plant physiologists associated with Philippe
`Anker and Maurice Stroun wasthefirst to characterize malig-
`nant features of CNAPSin cancerpatients [12]. This group had
`formerly investigated nucleic acid release from bacteria into
`plants (13), from frog auricles and from human lymphocytes [14].
`With another group [15], they further established the malignant
`nature of CNAPS in cancer patients by demonstrating gene
`mutations matching those of their primary tumors [16-17].
`Thus, as illustrated in cuRE 1, translating the general biologi-
`cal phenomenon of DNArelease from plant physiology to
`human oncology led to the establishment of a noninvasive
`molecular tumor detection method which hasresulted in more
`than 200 articles on the subject to date[7].
`Theinitial targeting of genomic tumor DNA in CNAPSanal-
`ysis has now been expanded tocirculating mitochondrial DNA,
`Thefirst step in CNAPS analysis consists of DNA isolation
`RNAand tumor-associated viral nucleic acids. The related use
`from plasma or serum. Careful
`two-step centrifugation has
`of CNAPSin nonmalignant indications [13], such as prenatal
`been recommended to ensure complete cell
`removal
`(28).
`diagnosis, transplant rejection and infectious diseases, will not
`Whereas higher absolute DNA concentrations have been
`be covered by this review. CNAPS have been found tobe parti-
`observed in serum [29], the relative proportion of tumor DNA
`tends to be higher in plasma [so]. 7 vitro release of benign
`cle-associated (see Methods). The use of quantitative detection
`methods for CNAPS has emerged rapidly since 1999 and has
`DNAfrom leukocytes during the coagulation process has been
`further increased the diagnostic utility of this method [19-23].
`proffered as an explanation for this observation [30,31]. There is
`no agreement on the most efficient method of DNAisolation
`However, despite favorable test characteristics compared with
`conventional serum tumor markers and other molecular detection
`from plasma/serum. The commercially available DNAisolation
`methods, so far CNAPSanalysis has notleft the research stage.
`kit from Qiagen (Venlo, The Netherlands) is widely used [31]
`Handling of the very small quantities of nucleic acids isolated
`and has compared favorably with manual isolation methods
`from plasmaor serum and dependence on comparably laborious
`32], although the superiority of that kit has been challenged by
`PCR amplification processes are regarded as the main obstacles
`other authors {33]. Quantities of CNAPS have been determined
`precluding this method from becomingclinical routine.
`using different methods yielding DNA concentrations of
`
`
`
`Genometastasis hypothesis
`A horizontal transfer of circulating tumor DNAinto tissues has
`been demonstrated in animal experiments and raises questions
`on the possible functional relevance of this so-called genome-
`tastasis, as opposed to conventional cellular metastatic spread
`24]. Hypothetically, under the assumption that horizontal
`transfer of tumor DNA sequenceshas a transforming potential
`[25,26], stem cells in distant organ tissues would constitute possible
`targets for uptake.
`
`The other plasma: cell-free tumor DNA in bone
`marrow supernatants
`According to a recent report by Taback and coworkers, tumor
`DNAdisplaying tumor-specific genetic alterations is present in
`the plasma (supernatant) of bone marrow aspirates from
`patients with early breast cancer [27]. The sensitivity associated
`with this approach was determined to be superior to analysis of
`circulating tumor DNA in the blood serum of the same
`patients. Due to its invasive nature, bone marrow analysis is not
`the focus of this review, although the work by Taback clearly
`demonstrates that analysis of cell-free tumor DNA begins to
`enter fields considered to be the domain of RNA-based detection
`of micrometastatic tumorcells {g}.
`
`Methods
`isolation of nucleic acids
`
`
`
`
`VW
`
`
`
`
`
`
`
`
`
`
`
`Bacteria [13]
`
`Lymphocytes [14]
`
`Tumor cells [12]
`
`Figure 1. Cellular DNA release: from bacteria to use as a molecular tumor marker in humans.
`
`432
`
`Expert Rev. Mol. Diagn. 3(4), (2003)
`
`00002
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`

`

`2-30 ng/ml in healthy controls (11.16,34-36] and 20-200 ng/ml
`in cancer patients [16,34-36]. However, absolute DNA amounts
`in plasma/serum do notreliably differentiate between cancer
`patients and controls, especially not in those with autoimmune
`diseases
`[11]. Fournie and coworkers found a correlation
`between advanced tumor stage and absolute DNA concentra-
`tions in lung cancer patients [35], however, no such associations
`were found in various other tumorentities including lung can-
`cer [31,34]. The relative proportion of tumor DNA within whole
`plasma/serum DNAis quite variable (38-93%) [36]. Colorectal
`cancer appears to be associated with comparatively low
`amountsofcirculating tumor DNAas evidenced by thefindings
`of Hibi and coworkers(37). Conversely, in many other tumor
`types, circulating tumor DNA appearsto be greatly enriched in
`the plasma/serum fraction [16,38]. The reasons for this phenome-
`non have been attributed to either increased release from malig-
`nant sources and/or reduced clearance from the circulation, the
`latter most
`likely attributable to its nucleoprotein structure
`(31,36,39]. The main bulk of DNA fragments in plasma/serum of
`cancer patients was found to be shorter than 200 bp (36). Bote-
`zatu and coworkers reported on short mutated 4-zas sequences
`secreted into the urine of cancer patients, indicating that at
`least short CNAPS might undergo renal filtration (40). If con-
`firmed in a larger series of different tumorentities, urine may
`emerge as a second source for universal molecular diagnosis of
`malignancy based uponcell-free methodology(18,41).
`Recently,cell-free circulating tumor RNAalso has been suc-
`cessfully isolated from plasma/serum of cancer patients [42-48].
`Given thesignificant RNAse activity of whole blood,this find-
`ing was unexpected. However, as demonstrated by Hasselmann
`and coworkers, circulating tumor RNA is embedded in apop-
`totic bodies and thus, as with circulating DNA,is protected
`against degradation. Although the exact mechanism of tumor
`DNAand RNArelease into the circulation remains unknown,
`there is accumulating evidence that most
`tumor-derived
`CNAPSare from cancer cells undergoing apoptosis (31,36,39].
`
`Circulating tumor DNA: target selection for molecular diagnosis
`DNAmutations, microsatellite alterations and epigenetic DNA
`alterations manifested by gene promoter hypermethylation
`constitute the three main targets for analyzing circulating
`tumor DNA in cancer patients. The rationale behind this
`approach is the expected occurrence of the same alterationsin
`primary tumortissue. However, most likely due to the existence
`of heterogeneous tumorclones (and/or undetected micrometa-
`static spread), alterations found in a small tumor sample do not
`always match those found in plasma/serum DNA [50-51]. Only
`the main features of the three most commonly used detection
`methodsfor circulating tumor DNA in plasma/serum of cancer
`patients will be presented.
`
`DNA mutation analysis
`Most investigators choose zas mutations when analyzing circu-
`lating tumor DNA. The existence of zas mutations in solid
`malignancies is especially common in cancers of the pancreas
`
`Diagnostic potential of circulating nucleic acids
`
`and gastrointestinal tract, where they occur in 90 and 50% of
`the primary tumors, respectively [7,52]. Since these mutations
`are clustered in codon 12 of the 4-ras gene, PCR-based meth-
`ods have been developed which enrich for common mutation
`variants in plasma/serum DNA [36.52]. However,
`the finding
`that 4-zas mutations can occur in benign conditions and thus
`appearto be not entirely tumor-specific, confers some concern
`about their usefulness as molecular tumor markers. In addition,
`Ramirez and coworkers found that 4-7as mutations in primary
`non-small cell lung carcinomasonlyrarely matched the consist-
`ent mutation pattern (i.e., TGT) in corresponding serum [53].
`Prospective, longitudinal studies are needed to eliminate specif-
`icity concernsassociated with this molecular target (54). Muta-
`tions of the tumor suppressor gene p53 have been investigated
`in plasma/serum of patients suffering from hepatocellular,
`breast and colorectal carcinoma [31], although the methodical
`workload associated with this approach does not favor routine
`clinical application.
`been
`have
`Recently, mitochondrial DNA mutations
`exploited as a target for analyzing plasma/serum DNA from
`cancer patients. Mitochondrial DNA is present in approxi-
`mately 200-10,000 copies per cell and should therefore provide
`a better sensitivity for detecting critically low quantities of tar-
`get DNA [81,55]. However, although three out of three prostate
`cancer patients with mitochondrial DNA mutations in their
`primary tumorexhibited the same mutations in serum [56], the
`detection rate in serum from ovarian cancer patients was 0%,
`perhaps due to the fact that no mutation enrichment method
`was applied [57]. However, even when using mutation enrich-
`ment PCR,the detection rate of tumor-derived mitochondrial
`DNAin colorectal cancer patients was disappointingly low
`(14% in primary tumors harboring target mitochondrial muta-
`tions) 58]. As mutations of genomic DNA (é-zas) can be
`detected in plasma/serum of more than 80% ofcolorectal cancer
`patients harboring the same mutations in the primary tumor
`(59), the high copy number of mitochondrial DNA does not
`compensate for decreased release efficacy and/or increased
`breakdownin plasma/serum ofcancerpatients.
`Results from different tumor entities with various disease
`stages are deemed necessary to better define the overall diagnostic
`sensitivity of this approach, irrespective of the problems asso-
`ciated with sequencing the whole or parts of the 16.5 kb
`mitochondrial genome.
`
`Microsatellite analysis
`Generally, microsatellite analysis is easy to perform, however, it
`requires tumor DNA within DNA originating from benign
`sourcesat a ratio of 0.5-5% when targeting microsatellite insta-
`bility (MIN) and more than 10-20% when targeting loss of
`heterozygosity (LOH) [16]. Since some tumorentities, such as
`bladder cancer, are not associated with consistent alterations at
`characteristic chromosomal locations, a panel of 17 markers
`was used by von Knobloch and coworkers for detection of
`tumor DNA in serum [60]. Despite the possibility of using
`multiplex PCR to reduce the workload, this level of complexity
`
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`

`Goessl
`
`Table 1. DNA alterations in plasma/serum of cancer patients (at diagnosis, all stages if not otherwise indicated).
`
`
`
`Alteration Patients (n)=Markers (n)Neoplasm Alterations in Specificity Ref.
`
`
`
`serum/plasma (%)
`(%)
`
`Microsatellite alterations
`
`LOH,
`
`LOH,
`
`LOH,
`
`LOH,
`
`LOH,
`
`LOH,
`
`LOH,
`
`LOH,
`
`LOH,
`
`LOH,
`
`LOH,
`
`MI
`
`MI
`
`MI
`
`MI
`
`MI
`
`MI
`
`MI
`
`MI
`
`MI
`
`MI
`
`MI
`
`on-small cell bronchial
`cancer
`
`Lung cancer
`
`eck/head tumors
`
`Colorectal cancer
`
`Colorectal cancer
`
`Clear-cell renal cancer
`
`Renal cell cancer
`
`elanoma
`
`Breast cancer
`
`Breast cancer
`
`Bladder cancer
`
`2)
`
`34
`
`2)
`
`44
`
`2/
`
`40
`
`40
`
`40
`
`2)
`
`5]
`
`39
`
`3
`
`6
`
`12
`
`8
`
`9
`
`4
`
`20
`
`10
`
`7]
`
`2
`
`17
`
`8
`
`71
`
`85
`
`29
`
`ot)
`
`59
`
`65
`
`8/
`
`58
`
`48
`
`30
`
`85
`
`93
`
`100
`
`100
`
`100
`
`nd
`
`nd
`
`100
`
`85
`(10 markers)
`
`100
`
`100
`
`100
`
`100
`
`100
`
`16]
`
`51]
`
`17]
`
`37]
`
`78]
`
`38]
`
`79]
`
`80]
`
`61]
`
`81]
`
`60]
`
`64]
`
`
`
`Ovarian cancer
`
`LOH
`allelic imbalance)
`
`Gene mutations
`
`54
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`N-ras
`
`Hematologic neoplasms
`
`Ig chain DNA
`
`B-cell leukemia
`
`K-ras
`
`k-ras
`
`k-ras
`
`k-ras
`
`Colorectal cancer$
`
`Colorectal cancer
`
`Colorectal cancer
`
`Pancreatic carcinoma
`
`10
`
`110
`
`8
`(adenoma: 62)
`
`14
`
`69
`(adenoma: 9)
`
`21
`
`50
`
`86
`
`63
`(adenoma: 35)
`
`50
`
`4]
`(adenoma: 44)
`
`81
`
`Nd
`
`100
`
`868
`
`100
`
`100
`
`100
`
`82]
`
`83]
`
`54]
`
`59]
`
`52]
`
`76]
`
`
`pd3
`Hepatocellular cancer
`20
`30
`100
`84]
`
`Gene promoter ypermethylatior®S
`p16, MGMT, GSTP1,
`on-small cell bronchial
`DAP kinase
`carcinoma
`
`p76 (Quant)
`
`Hepatocellular carcinoma
`
`pl6é
`
`p16, MGMT, GSTP1,
`DAP kinase
`
`Breast cancer
`
`eck/head tumors
`
`APC (Quant)
`
`APC (Quant)
`
`GSTP1
`
`GSTP7 (Quant)
`
`Esophageal cancer
`
`Lung cancer
`
`Prostate cancer
`
`Prostate cancer
`early stage)
`
`4
`
`4
`
`22
`
`22
`
`43
`
`50
`
`84
`
`89
`
`32
`
`69
`
`52
`
`59
`
`14
`
`42
`
`18
`
`4]
`
`72
`
`32
`
`Nd
`
`100
`
`100
`
`100
`
`100
`
`100
`
`100
`
`100
`
`85]
`
`23]
`
`86]
`
`87]
`
`20]
`
`21]
`
`88]
`
`22]
`
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`
`Expert Rev. Mol. Diagn. 3(4), (2003)
`
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`
`

`

`Diagnostic potential of circulating nucleic acids
`
`Table 1. DNA alterations in plasma/serum of cancer patients (at diagnosis, all stages if not otherwise indicated) (cont.).
`Alteration
`Neoplasm
`Patients (n)
`Markers (n)
`Alterations in
`Specificity
`Ref.
`
`serum/plasma (%)
` (%)
`
`Mitochondrial DNA mutations
`
`[89]
`
`Prostate cancer {early stage)
`
`Ovarian cancer
`
`Colorectal cancer
`
`Hepatocellular carcinoma
`
`3
`
`14
`
`77 (7885)
`
`1088
`
`OML
`
`Seq
`
`OMLSS
`
`OML
`
`100
`
`0
`
`1 (148%)
`
`goss
`
`[56]
`
`(57]
`
`[58]
`
`Nd
`
`Nd
`
`Nd
`
`Nd
`
`‘Prospective analysis: plasma DNA samples were taken before colonoscopy and pathohistological confirmation (screening situation).
`Scontrol group consisted mainly of symptomatic or high-risk patients. SSReview: [66]. “SSonly patients with confirmed mitochondrial mutationsin their primary tumor.
`APC: Adenomatouspolyposis coli gene; DAP: Death-associated protein; GSTP1: Glutathione-S-transferase P1 gene: Ig: Immunoglobulin; LOH: Loss of heterozygosity;
`MGMT. 06-methylquanine-DNA methyltransferase gene; MIN: Microsatellite instability; Nd: Not determined; OML: Oligonucleotide mismatch ligation assay
`
`(mutation enriching PCR technique); Quant: Quantitative determination using real-time methylation-specific PCR: Seq: Determination by sequencing serum DNA.
`
`raises concernsregarding the practicality of broad clinical appli-
`cation in CNAPSanalysis. In addition, microsatellite analysis
`using small DNA concentrations, such as those isolated from
`plasma/serum, is prone to technical artifacts [61-62]. Longeralle-
`les (>200 bp) are morelikely to display false-positive LOH than
`their corresponding shorter counterparts [63]. In CNAPSanaly-
`sis, the underlying difficulty to amplify longer microsatellite
`sequences can be ascribed to the highly fragmented nature of
`plasma/serum DNA [36]. Although true-positive LOH findings
`in plasma/serum DNAcan usually be confirmed by compari-
`son with findings in primary tumortissue and by use of addi-
`tional neighboring markers (38), specificity concerns, amongst
`others, are prohibiting broader routine clinical use of this
`method. According to Chang and coworkers,allelic imbalance
`in plasma/serum DNA can be analyzed using digital single-
`nucleotide polymorphism analysis without the potential risk of
`preferential amplification of shorter microsatellite alleles 64).
`This method is, however, associated with additional workload,
`leaving its suitability for routine CNAPSdiagnosis doubtful.
`
`Gene promoter hypermethylation analysis
`Gene promoter hypermethylation has becomeanattractive target
`for molecular cancer detection as promoter hypermethylation
`often constitutes an early neoplastic change, occurring even in
`premalignant or morphologically yet benign lesions [65). A
`comprehensive review on the use of gene promoter hypermeth-
`ylation analysis to detect exfoliated tumorcells in secreted body
`fluids as well as circulating tumor DNAin blood fractions from
`cancer patients was recently published by Laird [66]. Detection
`of
`tumor-associated gene promoter hypermethylation in
`plasma/serum DNA by methylation specific PCR (MSP) [67]
`significantly enhances the analytical sensitivity compared with
`microsatellite analysis. The methodical sensitivity of such assays
`equals approximately 1 x 104 {67} to 1 x 10° (6s). Since three
`methylatedsites are usually targeted by each of the corresponding
`two MSP-primers, false-positive results due to technical artifacts
`are not considered to be of concern. Age-related gene promoter
`hypermethylation (e.g., of APC and p/6 in normal gastric
`mucosa) is a well-known event with possible implications for
`
`analysis of plasma/serum DNA eo}. Fortunately, because normal
`tissues affected by gene promoter hypermethylation apparently
`do not secrete DNA into the blood circulation, this problem
`appears to be theoretical in nature as p/6 and APCgenepro-
`moter hypermethylation has not yet been observed in the
`serum/plasma of controls (20,66). To further protect against
`false-positive scoring, suitable cut-off levels can be defined
`when using quantitative MSP for tissue and/or plasma/serum
`samples [20-23]. Quantification of promoter hypermethylation
`provides a tool to distinguish low-level hypermethylation due
`to age-related processes and/or biological noise from high-level
`hypermethylation associated with neoplastic growth.
`
`Circulating tumor RNA
`The same rationale applies for analysis of circulating tumor
`cells as for analysis of circulating tumor RNA[5.6]. It is assumed
`that transcripts of epithelial origin do not normally occur in the
`blood, thus their existence is thought to indicate malignancy
`which has gained access to the circulation. This concept has
`been challenged bythe observationofillegitimate transcription;
`the secretion — albeit at low levels — of epithelial cell transcripts
`by any cell including normal blood cells and/or endothelial
`cells. Certain transcripts used as tumor targets are morelikely
`to be produced by normal nonepithelialcells (e.g., albumin and
`CK-19) than others whose occurrence is known to be more
`closely tumor-associated [69].
`
`Circulating viral nucleic acids
`In addition to tumor nucleic acids from endogenoussources,
`analysis of tumor-associated viral DNA has emerged rapidly as
`a moleculartool for initial diagnosis and follow-up monitoring
`in defined populations of cancer patients. Chan and coworkers
`found that patients with EBV-associated nasopharyngeal carci-
`noma had persistently elevated EBV-DNA levels in their
`plasma/serum, whereas patients after recovery from benign
`mononucleosis (caused by EBV) did not [19,70]. Quantitative
`determination of EBV-DNAalso yielded prognostic information
`and decreasing levels were strongly correlated with favorable
`response to therapy (70]. Determination of viral DNAlevels is
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`Goessl
`
`close to becoming routine application in these patients [78,70].
`The methodology mayalso haveclinical application potential
`in other virus-associated malignancies, such as EBV-associated
`lymphoma andgastric cancer [71.72], as well as Human Papil-
`loma virus (HPV)-associated cervical cancer and head and neck
`cancer [73-75]. A limitation to quantitative determination of
`viral DNAas part of CNAPSanalysislies in the fact that strong
`association with viral infection is limited to few cancer entities
`and to certain geographic areas (mostly Asia-Pacific region).
`
`Overview ofresults
`
`Circulating tumor DNA
`TABLE 1 provides data on detection rates of circulating tumor
`DNAinvariouscancer entities at initial diagnosis. This selection
`is incomplete for reasons of space. The detection rates are given
`for calculation of clinical sensitivity; prior finding of a target
`DNAalteration in the primary tumor was not considered a pre-
`requisite. A less conservative estimation, yielding numerically
`higher detection rates, is the calculation of methodicalsensitivity
`which, however, includes only patients with confirmed target
`DNAalterationsin their primary tumors [66].
`All the patients in TABLE1 were diagnosed using clinically
`established tumor detection methods and conventional patho-
`histological tumor confirmation was the gold standard against
`which positive CNAPS findings were compared. As different
`tumorstages were included in most studies, the table data do
`notspecifically address the question of how well CNAPSanalysis
`performsas a detection method specifically for early-stage cancer.
`In some but not all tumorentities, the finding ofcirculating
`
`tumor DNA mayindicate poor prognosis and/or advanced
`tumor stage [7.31]. However,
`the remarkable detection rate of
`80% &-zas mutations found by Kopreski and coworkers in
`plasma of patients with 4-zas positive colorectal adenoma pro-
`vides proof-of-principle evidence that CNAPSanalysis is able to
`detect early tumors, including preinvasive stages [54]. These find-
`ings have been recently confirmed by Ryan and coworkers who
`found 44% of patients with tubulovillous adenoma to beposi-
`tive for serum 4-zas mutations (57% when k-/as positive adeno-
`mas were selected) [52]. In contrast to this, molecular detection
`of circulating tumorcells is per definition dependent oninvasive
`growth. Thus, CNAPSanalysis has the potential to detect pre-
`malignancy and malignancyearlier than RT-PCR-based meth-
`ods for detection of circulating tumor cells @tGURE 2). In addi-
`tion, the early release and circulation of oncogenic nucleic acids
`from preinvasive malignancies could havea signal function for
`facilitating tumor growth both locally and at distantsites.
`The data in TABLE 1 demonstrate that CNAPSanalysis ofcir-
`culating tumor DNAis associated with a remarkable high spe-
`cificity, reaching 100% in mostcases. This is in sharp contrast
`to all pressently used protein-based serum tumor markers.
`However, in most studies on CNAPSin oncology indications,
`the numberof controls used was small and, in manycases, did
`not include inflammatory or other benign disease conditions.
`Data on using serial CNAPSanalysis for follow-up monitoring
`are even more sparse. In pancreatic, colorectal and small-cell
`lung cancer, longitudinal analysis of DNA alterations in the
`plasma/serum of patients was foundto be useful for monitoring
`during follow-up [52,76,771.
`
`
`
`Review
`reference
`
`[4-6,18,31,69]
`
`[4,7,8,69]
`
`
`
`
`
`
`
`Circulating tumor RNA
`TABLE 2 provides data oncell-free circulat-
`ing tumor RNA in cancer patients. In
`contrast to analysis of circulating tumor
`DNA,the targets are not alterations of
`nucleic acids but rather the mere detection
`of transcripts from epithelial origin is
`taken as a surrogate for malignancy in
`affected patients. In addition with telom-
`erase RNA, a tumor-specific RNA target
`has also become available for detection in
`plasma and serum of cancer patients
`[44,90]. Due to a certain rate of false-posi-
`tive findings in controls, specificity with
`some RNA markers(¢.g., CX-19) appears
`to be considerably less than 100% and
`lower than DNA-based detection methods
`in plasma/serum. Extensive comparisons
`between DNA- and RNA-based CNAPS
`analysis
`using
`identical plasma/serum
`samples from cancer patients and controls
`have, however, not yet been reported [69].
`Figure 2. Origin of circulating tumor DNA/RNAin the serum/plasma of cancer patients (CNAPS)
`There is only one report on CNAPSanal-
`as opposedto circulating tumor cells (CTC). Detection of malignant cell growth using CNAPS
`ysis comparingsensitivity of two markers
`analysis is possible even at preinvasive stages [52,54]. At present, the role of CNAPSasa cellular signal
`for cell-free RNA versus two microsatellite
`and its possible role in facilitating tumor spread and metastasis remains unknown [24-26].
`
`Tumor
`
`Blood/lymph vessel
`
`436
`
`Expert Rev. Mol. Diagn. 3(4), (2003)
`
`00006
`
`

`

`Diagnostic potential of circulating nucleic acids
`
`/{ Table 2. RNA ofepithelial origin in plasma/serum of cancer patients (at diagnosis, all stages).
`
` Target RNA Neoplasm Patients (n) Sensitivity (%) Specificity (%) Ref.
`
`
`
`
`Tyrosinase
`lalignant melanoma
`6
`67
`100
`42]
`
`Tyrosinase
`
`Telomerase
`
`Telomerase
`
`CK-19
`
`Mammaglobin
`
`CK-79 and mammaglobin
`
`CEA
`
`CK-19
`
`alignant melanoma
`
`Colorectal cancer
`Lymphoma
`
`Breast cancer
`
`Breast cancer
`
`Breast cancer
`
`Breast cancer
`
`Colorectal cancer
`
`Colorectal cancer
`
`Lung cancer
`
`10
`
`9
`9
`
`18
`
`45
`
`45
`
`45
`
`53
`
`53
`
`5
`
`60
`
`1008
`898
`
`25-285
`
`60
`
`49
`
`713
`
`32
`
`74
`
`Nd
`
`100
`
`100
`100%
`
`100
`
`80
`
`88
`
`Nd
`
`96
`
`80
`
`0
`
`43]
`
`90]
`
`44]
`
`45]
`
`45]
`
`46]
`
`47]
`
`47]
`
`48]
`
`
`
`
`
`
`CK-19
`
`HER2/neu + hnRNP-BT
`
`Lung cancer
`
`5
`
`100
`
`100
`
`48]
`
`SSensitivity and specificity obtained with a cut-off level higher than maximum valueobtained in ten healthy volunteers.
`SSDepending on telomerase subunit.
`
`CEA: Carcinoembryenic antigen; Nd: Not determined.
`
`markers for cell-free DNA [46]. The authors concluded that
`RNA-based analysis was superior to DNA-based analysis in
`plasma samples of breast cancer patients, however, specificity
`was not analyzed and, given the findings of Fleischhacker and
`coworkers (0% specificity for serum CA-/9RNA) [48], the over-
`all clinical superiority of analyzing cell-free RNA versus DNA
`in plasma/serum of cancer patients remains to be determined.
`Positive findings of circulating tumor mRNAwerecorrelated
`with poor prognosis in breast cancer and advanced-stage in
`colorectal cancer [46,47]. Furthermore, simultaneous analysis of a
`few transcripts from serum is already possible, thereby allowing
`a noninvasive miniprofiling of malignancy [48]. In addition,
`
`Dasi and coworkers reported on the possibility of quantifying
`plasmalevels of circulating tumor RNAin cancer patients {90}.
`They foundthatall nine patients suffering from lymphoma and
`eight out of nine patients suffering from colorectal cancer had
`higher plasma humantelomerase reverse transcriptase (47E27)
`mRNAlevels than the maximum value determined in ten
`healthy volunteers.
`
`Circulating viral DNA & RNA
`TABLE 3 Summarizes data on the use of viral DNA (and RNA) as
`a nucleic acid-based tumor marker. Quantitative analysis has
`further improved the diagnostic specificity for initial diagnosis
`
`/ Table 3. Circulating viral DNA (and RNA) in plasma/serum of cancer patients (at diagnosis, all stages).
`Target
`Neoplasm
`Patients (n)
`Sensitivity (%)
`Specificity (%)
`Ref.
`EBV-DNA
`asopharyngeal cancer
`42
`3]
`100
`91]
`
`
`
`
`
`
`EBV-DNAS®
`
`EBV-RNA
`
`EBV-DNAS
`
`EBV-DNA
`HPV-DNA
`
`HPV-DNA
`
`asopharyngeal cancer
`
`asopharyngeal cancer
`
`EBV-associated T-cell lymphoma
`
`EBV-associated gastric cancer
`Cervical cancer
`
`Cervical cancer
`Cervical cancer in situ
`
`Head and neck squamous cancer
`
`HPV-DNA
`SQuantitative PCR
`‘Controls: detection rate in gastritis patients 23%: in healthy controls 3.6%.
`FBV: Epstein-Barr virus; HPV: Human papilloma virus; Nd: Not determined.
`
`57
`
`26
`
`18
`
`19
`50
`
`175
`57
`
`10
`
`96
`
`89
`
`94
`
`93-100
`12
`
`7
`2
`
`6-9
`
`93
`
`19
`
`100
`
`77-968
`100
`
`98
`
`Nd
`
`19]
`
`92]
`
`71]
`
`72|
`73]
`
`74]
`
`75]
`
`www.future-drugs.com
`
`437
`
`00007
`
`

`

`target RNAs may become amenable to noninvasive analysis
`using the low amounts of RNApresent in plasma/serum [48].
`In addition, analyzing DNA mutation and gene promoter
`methylation patterns also has a potential for profiling tumors
`yielding prognostic and/or predictive (response to therapy)
`information (67,99-101). The information value may be further
`increased when using quantitative approaches in CNAPS
`analysis
`[19-23]. Response to chemotherapy appears to be
`reflected by RNA expression changes [102] and — especially in
`the case of agents evoking demethylation and histone acetyla-
`tion - also by altered gene promoter methylation patterns
`(86,69,103]. It is expected that within 5 years, these responses at
`the DNA methylation and RNA expression levels will be
`determined noninvasively by quantitative CNAPS analysis
`(86,68,90,103]). Implementing additional promising techniques,
`such altered serum proteomic pattern analysis [94], into such a
`setting could result in a quick and feasible surrogate for drug
`response, without the need to obtain repeated biopsies. Thus,
`CNAPSprofiling may becomea valuable tool for speeding up
`drug development processes. Most importantly, by enabling
`physiciansto tailor systemic therapy to individual patient reac-
`tions, CNAPSprofiling may finally result in a better and
`longer life for cancer patients.
`
`Key issues
`
`Circulating nucleic acids in plasma and serum (CNAPS)
`analysis has emerged as a noninvasive tool for initial
`detection of malignancy as well as an indicator of recurrence
`uring follow-up.
`oth tumor DNA and RNA can be detected in plasma/serum
`f cancer patients.
`mited tumorprofile yielding prognostic and/or predictive
`esponse to therapy) information is already possible with
`APSanalysis.
`The method needs validation by performing PhaseIII clinical
`rials focusing on higher patient and control numbers, early
`disease stages and different control groups including
`inflammatory diseases.
`Quantitative determination of viral DNA in virus-associated
`malignancies, such as nasopharyngeal carcinoma, is close to
`becoming routine application for initial diagnosis and
`monitoring during follow-up.
`t is expected that future ro