Clinical Biochemistry 37 (2004) 605 617

Review

Apoptotic markers in cancer

S. Holdenrieder and P. Stieber *
Institute of Clinical Chemistry, University Hospital Munich-Grosshadern, D-81377 Munich, Germany Accepted 4 May 2004

Abstract In cancer, apoptotic processes occur both spontaneously and induced by antitumor therapies. Qualitative and quantitative changes in cancer cell death along with proliferative alterations are essential determinants in the pathogenesis and progression of malignant disease and its responsiveness to therapy. Besides detecting apoptosis by invasive means in tumor tissue, apoptotic products can be quantified in the circulation. Although circulating apoptotic products usually lack organ and tumor specificity, they contribute in the assessment of disease extent or aggressiveness. The ease of drawing blood facilitates the serial measurement of circulating apoptotic markers to monitor antitumor treatment and predict early response to therapy. This review describes the features of apoptotic and necrotic cell death along with the role the balance between the rates of cell death and cell proliferation plays in the progression of malignancy. The intracellular pathways mediating apoptosis are next summarized. The focus then shifts to the apoptotic markers found in the circulation and their diagnostic, prognostic, predictive, and management utility in cancer. D 2004 The Canadian Society of Clinical Chemists. All rights reserved.
Keywords: Cell death; Apoptosis; Cancer; Tumor; Diagnosis; Prognosis; Therapy; Monitoring; Prediction; sFas; CD95; Cytokeratins; CYFRA 21-1; TPA; TPS; DNA; Nucleosomes

History of cell death Although studies on cell death were reported to be performed by Aristotle and later by Galen, who described the regression of larval and fetal structures during ontogenesis, it took until the 19th century for cell death processes to gain the interest of pathologists [1,2]. In 1842, Carl Vogt [3] discovered without the benefit of histochemical methods that cells can undergo programmed cell death. Further specifications and definitions of cell death were introduced by Virchow in 1858 (e.g., degeneration, softening, necrosis, mortification) [1,2], by Flemming in 1885 (spontaneous cell death, chromatolysis) [4], and by Graeper in 1914 (chromatolysis as a counterpart to mitosis) [5]. After World War II, embryologists continued the research on cellular demise and characterized the degradation of embryonic tissue as a result of single cell death [6]. In 1971, John F. Kerr [7] observed specific alterations of dying liver cells after occlusion of the vena portae in a rat model that he named shrinkage necrosis. One year later, he introduced with Andrew Wyllie and Sir Alastair Currie the term apoptosis (Greek: falling of the leaves) to describe this phenomenon and founded the field of modern cell death research [8].

Features of cell death Apoptosis typically affects single cells that are aged, dysfunctional, or damaged by external stimuli. It is an active, energy-requiring process leading to a well-regulated degradation of the cell. Early pathomorphological features are chromatin condensation and marginalization in the nucleus, DNA fragmentation into mono- and oligonucleosomal units, cellular shrinkage, karyorrhexis, packing of organelles, and dilatation of the endoplasmatic reticulum. Initially, mitochondria, lysosomes, and cellular membranes remain functionally intact. Later, budding of cellular membrane is observed leading to the packaging of cellular components into vesicles that are released as apoptotic bodies and phagocytyzed by macrophages and neighboring cells [1,9,10]. Loss of phospholipid asymmetry in the plasma membrane plays an important role in the opsoniza-

* Corresponding author. Institute of Clinical Chemistry, University Hospital Munich-Grosshadern, Marchioninistr. 15, D-81377 Munich, Germany. Fax: +49-89-7095-6298. E-mail address: Petra.Stieber@med.uni-muenchen.de (P. Stieber).

0009-9120/$ - see front matter D 2004 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2004.05.003

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tion of apoptotic bodies and subsequent phagocytosis [11 13]. Under physiologic conditions, a well-organized recycling system regulates the recognition, incorporation, and lysosomal ingestion of the cellular remnants avoiding inflammatory reactions in the tissue [8,10,14]. The early apoptotic events occur within minutes while the final stages of lysosomal degradation are finished within hours, the time course depending on cell type and tissue [1,9,10,15]. Necrosis characteristically affects groups of cells or whole tissue after extended damage induced by external stimuli like trauma, ischemia, and high dose irradiation. Loss of function of mitochondria and endoplasmatic reticulum leads to a dramatic breakdown of energy supply resulting in cellular, nuclear, and organellar swelling, blebbing of the membrane, and final rupture of the plasma membrane with release of lysosomal enzymes into the extracellular space. Neighboring cells and tissues in the vicinity are attacked by released proteases leading to extended inflammatory reactions [10]. In the nucleus, chromatin flocculation and nonspecific degradation of DNA by proteases is observed [16,17]. The whole process of necrosis is terminated within 12 24 h [1]. Characteristic features of apoptosis and necrosis are summarized in Table 1.
Table 1 Morphological and biochemical characteristics of apoptosis and necrosis Apoptosis Prevalence Localization Grade of activity Pathomechanism Affects single cells Margins of lesions or single cells Active, energy requiring ATP-dependent activation of proteases and endonucleases Loss of water Shrinkage Shrinkage Karyorrhexis Loss of nucleoli Nuclear condensation and marginalization Fragmentation into pieces of 50-300 kbp Fragmentation in multiples of 180 bp by endonucleases Functional Structurally intact Decreased transmembrane potential Budding Packaging of cellular content into membranederived vesicles Apoptotic bodies Phagocytosis by neighboring cells and tissue macrophages No Necrosis Affects groups of cells Center of extended lesions Passive, energy independent Dysfunctional ubiquitin system Dysfunctional ion pump Influx of sodium and water Swelling Swelling

Cell Nucleus

Chromatin

Flocculation Nonspecific fragmentation

Organelles Mitochondria

Membrane

Elimination

Dysfunctional Swelling Dysfunctional ubiquitin system Blebbing Loss of impermeability Rupture Release of cellular content Migration of macrophages Yes

Inflammation

In some cases, apoptosis and necrosis cannot be unambiguously distinguished by these characteristics. For example, cells with morphological necrotic features may show specific DNA fragmentation [18,19], while processes with apoptotic morphology may occur lacking the typical DNA fragmentation [19 21]. A variety of cell death modalities have been described that are intermediate in nature and appearance between necrosis and apoptosis [17,22 25]. As they were observed after both internal and external induction of cell death, it is hypothesized that the severity more than the mode of the stimuli influences the manner of cellular demise [1,17,24,26]. Other influencing factors are the cell type and its predilection for a certain mode of cell death. Whereas cells with high energy levels choose active apoptosis, those with a low energetic level frequently will undergo passive, necrotic cell death [27 29]. The intracellular calcium concentration and pH are also believed to play a role [30,31]. In respect to terminology of cell death, a clarification seems to be reasonable. Often, the terms apoptosis and programmed cell death are used incorrectly as synonymsprobably as the result of confounding two programs that contribute to physiological cell death: (A) a trigger program regulating the time point of cellular suicide, and (B) an execution program regulating the mode of cellular degradation. Programmed cell death describes the genetic triggering of cell death without any specification of the mode of cell death (program A). In contrast, apoptosis is defined as a cell death modality characterized by specific morphological and biochemical features that can be induced by genetic or external stimuli (program B). Depending on the trigger and the mode of cell death, programmed cell death and apoptosis can occur along with or apart from each other while indicating distinct parts of physiological cell death [1]. The temporal disconnect between morphological and biochemical changes further complicates establishing the exact mode of cell death. Processes leading to cell death and those that are the consequences have to be discriminated. In many cases, dying cells reach a point of no return early after the onset of a lethal stimulus. However, the morphological features become evident only hours later. Irreversible changes of the nucleus and cellular plasma characteristic of necrosis take place mainly postmortem [1,15,17]. These aspects have been integrated by Majno and Joris [1] in a new concept of cell death: oncosis (cellular swelling) and apoptosis (cellular shrinkage) are considered to be opposing modalities of cell death while necrosis is restricted to refer to morphological postmortem changes. However, additional forms of cell death like autophagic cell death, paraptosis, and cytoplasmatic cell death add further complexity [22,23]. A global view of cell death processes induced by physiological (intrinsic) and accidental (extrinsic) stimuli must take into account that apoptosis and oncosis coexist with varying mixtures of the

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Fig. 1. Integrated cell death model indicating the complex interaction of cell death stimulus, trigger, modality, and postmortem status.

two observed. An integrated model of cell death modalities is shown in Fig. 1.

Cell death in physiological and pathological processes Homeostasis of cell proliferation and cell death plays an essential role in the maintenance of the function of organs and organisms [10,32]. These processes are characteristically unbalanced without adverse consequences during embryonic development, during establishment of immunologic tolerance, and during regeneration [33 39]. However, imbalance can also lead to adverse consequences such as the clonal expansion of malignancy when cells become resistant to apoptosis or proliferate excessively. Conversely, excessive cell death and/or diminished proliferation may be deleterious. Such imbalance occurs in degenerative diseases like AIDS, ischemia, toxin-mediated or autoimmune diseases, and in some types of cancer [34,38,40,41]. Cell death in cancer A more detailed view reveals the complexity of the processes leading to dyshomeostasis. The relative rates of cell death and cell proliferation may vary as malignancy progresses [38,42,43]. Initially, cell death may increase in parallel to cell proliferation, thereby leading to no net increase in cellular number. Should cell death mechanisms become attenuated, tumor will grow and invade local structures. Angiogenesis when induced will further support the rate of tumor proliferation. Concurrently, cell death could be further lessened or even enhanced. At later stages in the course of the malignancy, the rate of cell death may increase mirroring the presence of evermore dysfunctional cells [44,45]. In patients with malignant tumors, therapeutic strategies aim at equilibrating the imbalance between proliferation and degeneration. Depending on type and dosage of the chemotherapeutic drugs, the modality of radiotherapy, and the sensitivity of the tissue, cellular damage mostly results in arrest of the cell cycle and in response to insufficient repair to induction of active apoptotic cell death [46 49]. Quantifying cell death and cellular proliferation can provide information about the process of carcinogenesis

and the response to antitumor treatment. During physiological apoptosis, most of the cell death products are effectively removed by macrophages and neighboring cells and can be found in the circulation only in small amounts [10,13,50]. However, if the recycling system is impaired or overloaded by the malignant pathological condition, cell death products can accumulate in appreciable quantity in the circulation [51 53] and become potentially useful in the diagnosis, prognosis, and monitoring of disease. As many factors are involved in the process of cell death, it is crucial to choose the most cancer- and organ-specific biomarkers for these purposes. Further challenges are the stability of these factors in vivo and in vitro in serum or plasma, the confounding effects of renal insufficiency, infections, and hepatic failure on biomarker concentration, and the transient appearance of single markers and staggered temporal pattern of panels of multiple markers.

Factors of cell death Generally, intracellular components that are not secreted physiologically and appear in the extracellular space only after disintegration of the plasma membrane are suited for the quantification of cell death. Factors specifically taking part in apoptotic processes could be expected to deliver useful information regarding the mode of cell death. Apoptotic markers are involved at various levels. Receptors on the surface accept the death signal and transmit it across the cellular membrane. Specific intracellular enzymes are activated in order to coordinate the well-orchestrated breakdown of cellular structures. Sets of inhibitors and enhancers modulate the apoptotic signal. Finally, specific death substrates are degraded and externalized in a defined way. Receptors Various receptors are known to transmit the apoptotic signal. Most of them are members of the TNF family, including Fas/Apo1/CD95, TNFR1, DR3, DR4, and DR5. These receptors are glycosylated transmembrane proteins

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consisting of an extracellular component of two to six cysteine rich domains and an intracellular part containing death domains. After binding of specific ligands or stimulating antibodies, the receptors are activated by trimerization that promotes the association of intracellular proteins with the death domains to form the death-inducing signaling complex (DISC) [35,54 56]. Some receptors like Fas/Apo1/CD95 transduce the signal directly to specific apoptotic effector caspases, while others like TNFR1 use alternative intracellular pathways via NF nB and JNK/AP-1,

which are also involved in immune response, differentiation, and proliferation [54,55]. Enzymes and pathways The intracellular transmission of the apoptotic signal is regulated by a well-organized system of initiator and effector caspases (cysteine-dependent aspartate-specific proteases). These proteases activate further procaspases by cutting specifically after an aspartate sequence. Initia-

Fig. 2. Summary of apoptotic pathways illustrating receptor activation, formation of the death-inducing signaling complex (DISC), and activation of initiator ). Alternatively to the direct involvement of the caspase cascade, the signal caspases like caspase-8 and of downstream effector caspases like caspase-3 ( can be enhanced by an amplification circle including mitochondrial release of cytochrome c and activation of caspase-9 in the apoptosome or induce additional mechanisms like the release of the apoptosis-inducing factor (AIF). All pathways merge in the degradation of cell death substrates like cytokeratins and nucleosomal fragmentation of chromatin. Inhibitors ( ) regulate various steps of the cascade and enable fine-tuning of the apoptosis process (see text).

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tor procaspases bind to the DISC where they are autocatalytically transformed into their active form. After a cascade of proteolytic activation of further proteases, the signal finally reaches the effector caspases, which attack defined cellular molecules leading to the typical biochemical and morphological features of apoptosis [47,56,57]. With respect to the Fas/Apo1/CD95-receptor-mediated induction of apoptosis, the formation of the DISC and the activation of effector caspases are observed in all kinds of cells. However, differences occur in (1) the direct activation of the caspase cascade, (2) the involvement of the mitochondria and the release of cytochrome c, and (3) the release of apoptosis-inducing factor by the mitochondria [56,58,59]. Generally, apoptosis can proceed: (a) With rapid formation of the DISC and strong activation of initiator caspase 8, which is able to activate directly the caspase cascade including effector caspase 3 as well as mitochondria. (b) With slow DISC formation and weak activation of caspase 8. In this case, the apoptotic process requires the further activation of mitochondria by the molecule, BID, which results in the release of cytochrome c, cytoplasmatic aggregation of cytochrome c with apoptosis activating factor 1 (APAF-1), ATP and procaspase 9 in the apoptosome, and subsequent activation of the caspases 3 and 8. Caspase 8 further activates BID, thereby establishing an amplifying loop in the apoptoticsignaling pathway [56,58,59]. (c) With release by the mitochondria of apoptosis-inducing factor (AIF), which is capable of provoking morphologic characteristics of apoptosis in conjunction with and independently of the activation of caspases [60].

Inhibitors All essential steps of these pathways are regulated by inhibitors and enhancers that modulate the apoptotic process according to the demands and the possibilities of specific cells. Due to its central position in the transduction of the apoptotic signal, DISC is an important modulation site. FADD-like ICE inhibitory proteins (FLIPs) compete with procaspase 8 for the binding sites at death effector domains on the DISC and have been reported to inappropriately impair apoptosis in tumor cells and in virus-infected cells [58,64]. Other sites of modulation include the initiator caspases, the mitochondrial pathway, and the effector caspases themselves. Cytokine response modifier A (CrmA) effectively blocks caspases 1 and 8 [65 67]. Members of the bcl 2 family inhibit the stimulation of mitochondria by proapoptotic Bax, the release of cytochrome c and AIF into cytoplasm, and the recruiting of procaspase 9 to the apoptosome [34,68 70]. Inhibitors of effector caspases are named inhibitors of apoptosis (IAPs) and include survivin, XIAP, NAIP, and c-IAP-1 and 2. They bind to caspases 3, 7, and to the apoptosome and exhibit significant differences in their inhibiting potential [71 74], enabling effective finetuning of the apoptotic signal. This system is further optimized by IAP-regulating proteins like second mitochondria-derived activator of caspases (Smac) and direct IAPbinding protein with low pI (DIABLO) [47,75,76]. The most important components of the apoptotic pathways are illustrated in Fig. 2.

Methods of detecting cell death Many qualitative and quantitative methods are available to detect cell death by assaying the cell itself and through identifying apoptotic products released into the circulation (Table 2). Cellular- and serum-based tests show significant differences in sensitivity, specificity, and cost effectiveness. Originally, morphologic observations led to the characterization of various types of cell death [8,14]. Additional use of dyes enabled reliable quantitative determinations as well as functional tests of the plasma membrane and organelles. More elegant options for high throughput investigations were established by cytometry and fluorescent markers, which enable the interpretation of volume, structural, and functional changes such as the maintenance of phospholipid asymmetry in the plasma membrane. Further techniques detect cell-death-specific fragmentation of chromatin by DNA electrophoresis, cytometry, filtration assays, quantitative real-time PCR, and recognition of DNA breaks with various fluorescent markers. Finally, improved understanding of the regulating processes and signal pathways involved in cell death has led to new tests that measure the activity of caspases and the presence of specific substrates [17,77,78].

Substrates The various pathways of intracellular signal transduction merge into the activation of effector caspases with caspase 3 playing a particularly key role. These caspases (1) cleave structural components of the cytoskeleton and the nuclear membrane like actin, cytokeratins, and lamins; (2) cause phosphatidylserine to be exposed on the outside of the cellular membrane promoting phagocytation by macrophages and neighboring cells; (3) counteract the apoptosis-inhibiting effect of Bcl 2 und Bcl-xL proteins; (4) inhibit genes that regulate the repair of DNA lesions during cell cycle like mdm-2 and the Rb-gene; (5) inactivate enzymes responsible for stability, integrity, and repair of DNA, like poly-(ADP-ribose) polymerase PARP, DNAPK, and DNA replication factor 140; and (6) cleave ICAD (inhibitor of the caspase-activated DNase) and activate CAD (caspase-activated DNase), which causes the characteristic oligonucleosomal fragmentation of the chromatin [34,61 63].

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Table 2 Methods for detection of apoptosis at cellular level and in blood Methods Cellular level Morphology Electron microscopy Light microscopy Cytometry Light scatter Integrity of plasma membrane Uptake of dyes Release of cellular enzymes Function of organelles AnnexinV-phosphatidylserine DNA fragmentation Agarose gel electrophoresis Fractioning Filtration Inverse field gel electrophoresis TUNEL ISEL Cytometry Polymerase chain reaction Activity of enzymes Inhibition of enzymes Cell death products Serum and plasma Receptors and ligands Components of cytoskeleton Intracellular enzymes Components of nucleus Cell death products and regulators Examples Qualitative Quantitative Cost

+++ + ++ + Trypane blue, propidium, iodide, FDA LDH, adenylate cyclase Rhodamine 123, acridine orange + + + ++ + + + + ++ ++ ++ +++ ++ ++ ++ + + + + +

+ ++ ++ ++ ++ ++ + ++ + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + ++ + + ++ ++ ++ ++ ++ ++

Caspases, DNases ATA, CMK, FMK PARP, cytokeratins, nucleosomes Fas, FasL Cytokeratins, lamins NSE, LDH Matrix proteins, DNA, nucleosomes Caspases, cytochrome c, APAF1, PARP, ICAD, DNA, nucleosomes, survivin, IAPs, Bcl 2, p53

Qualitative and quantitative potential of the methods are indicated gradually ( low; + intermediate; ++ good; +++ very good), similarly costs ( very expensive; + expensive; ++ intermediate; +++ low priced).

These components and products of cell death can also be measured in serum and plasma, as well as in other body fluids like urine, cerebrospinal fluid, pleural effusions, and ascites. In addition, assays have been developed that detect and quantify apoptosis receptors and their ligands, elements of the cytoskeleton including cytokeratins and lamins, intracellular enzymes like LDH and NSE, free DNA and nucleosomes originating from the nucleus, caspases, their enhancers and inhibitors, and substrate breakdown products. Though blood investigations often lack organ and cell death specificity, because blood is easily drawn, circulating markers lend themselves to serial measurement that aids in monitoring the course of disease management.

sFas and its ligand FasL, cytokeratins, and circulating DNA fragments. Although several assays exist to measure caspase activity, to quantify substrates like PARP, and to determine inhibitors of apoptosis proteins (IAPs) like survivin [79,80], little has been published regarding the clinical utility of circulating levels in cancer. Fas-receptor and Fas-ligand Primarily, it has been the antiapoptotic soluble form of the fas-receptor (sFas) and the proapoptotic fas-ligand (FasL) that have been investigated in serum (Table 3). In contrast to healthy persons, patients with various kinds of cancer showed elevated levels of sFas in colon cancer [81], gastric cancer [82], liver cancer [83], breast cancer [84,85], gynecological cancers [86,87], renal cancer [88,89], melanoma [90,91], and non-Hodgkins lymphoma [92]. In some of these tumor types, sFas correlated with tumor stage [82,84,86,88,91] and with progressive disease [93]. However, several studies have reported that sFas is not elevated in cancer [94,95] and not correlated to stage [94,96]. Soluble Fas ligand (sFasL) has been reported to be elevated in gastric cancer [97], bladder cancer [98], and melanoma [90] and to have an association with tumor stage [98].

Circulating apoptotic markers in cancer Among the various apoptosis assays in blood, several have been evaluated clinically to determine their usefulness for diagnosis, prognosis, therapy monitoring, therapy prediction, and detection of recurrent disease in patients with cancer disease. The most frequent markers investigated in cancer patients have been the soluble forms of the apoptosis receptor

S. Holdenrieder, P. Stieber / Clinical Biochemistry 37 (2004) 605617 Table 3 Studies on soluble Fas receptor and Fas ligand Author Kushlinskii et al. [81] Liang et al. [82] Peng et al. [83] Sheen-Chen et al. [84] Nonomura et al. [88] Mouawad et al. [90] Pignataro et al. [95] Ichikura et al. [97] Ueno et al. [85] Munker et al. [94] Osorio et al. [93] Marker sFas sFas sFas sFas sFas sFas, sFasL sFas, sFasL sFasL sFas sFas sFas Cancer Colon cancer Gastric cancer HCC Breast cancer Renal cancer Melanoma Laryngeal cancer Gastric cancer Breast cancer NHL B-CLL n 33 42 36 57 31 45 26 112 233 66 83 Control Healthy Healthy Liver cirrhosis Benign Healthy Healthy Healthy Healthy Healthy Healthy Healthy 34 35 155 118 20 21 n 33 30 49 12 Results elevated in cancer elevated in cancer elevated in cancer elevated in cancer elevated in cancer elevated in cancer not elevated in cancer elevated in cancer elevated in cancer not elevated in cancer elevated in progressive disease elevated in cancer elevated in cancer elevated in cancer elevated in cancer elevated in cancer elevated in cancer not elevated in cancer elevated in cancer Correlation with stage no yes Prognosis univariate Prognosis multivariate

611

yes yes

no no yes

high sFas poor PFS and OS no correlation with PFS high sFas poor OS

sFas independent for PFS and OS

sFas not independent

Konno et al. [86] Hara et al. [96] Niitsu et al. [92] Kimura et al. [89] Ugurel et al. [91] Hefler et al. [87] Tsutsumi et al. [101] Mizutani et al. [98] Bewick et al. [99] Mizutani et al. [100] Kanda et al. [102]

sFas sFas sFas sFas sFas sFas sFasL sFasL sFas sFas, sFasL sFasL

Gynecological cancers NHL NHL Renal cancer Melanoma Ovarian cancer Gastric cancer Bladder cancer Breast cancer Bladder cancer Multiple Myeloma

64 67

Healthy Healthy Healthy

24 36

yes no

high sFAS poor OS high sFAS poor OS high sFAS poor OS high sFAS poor OS

sFas independent for OS

72 125 52 166 163 94

Healthy Healthy Healthy, benign Healthy Healthy

17 30 65 43

yes

high sFas poor PFS and OS high sFas poor PFS and OS high sFasL poor OS high sFasL poor PFS high sFas poor PFS and OS high sFas and sFasL poor PFS no

sFas independent for OS sFas independent for OS sFas independent for PFS not for OS sFas independent for PFS and OS sFasL independent for OS

yes

sFas independent for PFS and OS

35

no

Summary of studies dealing on the diagnostic and prognostic value of soluble Fas receptor (sFas) and Fas ligand (sFasL) illustrating the markers investigated, type of cancer diseases, control groups, number of patients (n), diagnostic results, correlation with stage, univariate and multivariate prognostic results concerning overall survival (OS), and progression-free survival (PFS).

The prognostic value of both markers has been investigated in a variety of cancers. In univariate analyses, high sFas levels were related to poor overall survival (OS) in breast cancer [85,99], gynecological cancers [86,87], melanoma [91], renal cancer [89], non-Hodgkins lymphoma [92,96], and B-CLL [93] and associated with poor progression-free survival (PFS) in breast cancer [85,99], ovarian cancer [87], bladder cancer [100], and melanoma [91]. sFasL revealed prognostic information for PFS in bladder cancer [98,100] and gastric cancer [101]. Other studies report no prognostic value for sFas in non-Hodgkins

lymphoma [94] and for sFasL in multiple myeloma [102]. In multivariate analyses, sFas proved to be an independent prognostic factor only for breast cancer [85,99], gynecological cancers [86,87], melanoma [91], renal cancer [89], and non-Hodgkins lymphoma [92]. Cytokeratins Cytokeratins are intracellular structural proteins quite specific to epithelial cells that are cleaved and released during apoptosis [103 105]. Although their soluble frag-

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ments are not tumor specific, they have been quantified in a number of studies to establish their utility in tumor diagnosis, prognosis, therapy monitoring, and detection of recurrent disease. The fragments measured have included CYFRA 21-1 (cytokeratin 19-fragments), which is the most widely used and specific of the cytokeratin markers in clinical practice [106 108], tissue polypeptide antigen (TPA, cytokeratin 8-, 18-, and 19-fragments), and tissue polypeptide-specific antigen (TPS, cytokeratin 18-fragments) [103]. Recently, a new cytokeratin fragment specific to early apoptosis has been identified [109]. It arises from cleavage of cytokeratin 18 by caspase 8. Such cleavage unmasks an epitope that is specifically detected by the M30-antibody. Further studies suggest that different portions of circulating cytokeratin 18 may be useful
Table 4 Studies on circulating DNA fragments Author Leon et al. [157] Marker DNA Cancer Various cancers GI cancers Lung cancer Lung cancer Various cancers Breast cancer Various cancers Various cancers Breast cancer n 173 199 45 68 185 96 220 418 125 Control Healthy n

to distinguish between apoptotic and necrotic processes [110]. Circulating levels of CYFRA 21-1, TPA, and TPS show some association with tumor stage and the rate of cancer cell lysis during therapy. Diagnostic and differential diagnostic value have been demonstrated in squamous cell cancers of the lung and the bladder with CYFRA 21-1 exhibiting the best profile of sensitivity and specificity [111 117]. Serum elevation has also been reported in small cell lung cancer, cervical, nasopharyngeal, esophageal, gastric, cholangiocellular, liver, colorectal, breast, and ovarian cancer [118 134]. Nonmalignant causes of cytokeratin elevations include renal insufficiency and severe infections with high rates of cell death [107,108,117,135,136]. Prognostic value of CYFRA 21-1 has been found in nonsmall cell and small

Results

Correlation Prognosis with stage yes

Therapy n RT

Monitoring therapy

Shapiro et al. [158] DNA Maebo [159] DNA

Fournie et al. [160] DNA Holdenrieder et al. [162] Kuroi et al. [161] Holdenrieder et al. [166] Holdenrieder et al. [165] Kuroi et al. [164] Nucleosomes Nucleosomes Nucleosomes Nucleosomes Nucleosomes

Sozzi et al. [167] Trejo-Becerril et al. [169] Sozzi et al. [171] Silva et al. [177] Lo et al. [163] Lo et al. [180]

DNA

Lung cancer Nucleosomes Cervical cancer DNA Lung cancer DNA Breast cancer EBV-DNA Nasopharyngeal cancer EBV-DNA Nasopharyngeal cancer EBV-DNA EBV-DNA

81 11 100 147 57 25

55 elevated in cancer Healthy, 275 elevated benign in cancer Healthy, 113 elevated benign in cancer Healthy elevated in cancer Healthy, 80 elevated infections in cancer Healthy 111 elevated in cancer Healthy, 90 elevated infections in cancer Healthy, 172 elevated benign in cancer Healthy, 212 elevated in cancer benign, other ca Healthy 32 elevated in cancer Healthy 14 elevated in cancer Healthy 100 elevated in cancer Healthy 35 elevated in cancer Healthy 43 elevated in cancer elevated in recurrent cancer

173 yes

yes yes yes (univariately) CT, RT no yes CT, RT no CT 36 yes, early peak 18 yes, early peak 38 yes 11 yes, early peak 35 yes no (only tendency) 24 yes, early peak

no

no

FU CT

no yes (univariately) yes

FU

RT FU

15 yes 17 yes

Lo et al. [181] Lo et al. [176]

Nasopharyngeal 10 cancer Nasopharyngeal 91, 139 cancer Lymphoma 26 Healthy

RT elevated in early recur. cancer 35 elevated in cancer yes (uni- or multivariately) yes (univariately) CT, RT

10 early peak

Lei et al. [178]

EBV-DNA

15 yes

Summary of studies dealing on the value of circulating DNA fragments for diagnosis, prognosis, and therapy monitoring. Markers investigated, type of cancer diseases, control groups, number of patients (n), diagnostic results, correlation with stage, prognostic results, kinetic investigations during therapies (CT = chemotherapy, RT = radiotherapy, FU = follow-up), and results of therapy monitoring are indicated.

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cell cancer of the lung [137 141], mesothelioma [142], esophageal cancer [143], and cervical cancer [144]. CYFRA 21-1 has additional proven utility in monitoring treatment in lung cancer [145,146], head and neck cancer [147,148], and cervical cancer [149], in predicting response to therapy in lung cancer [146,150,151], and in predicting recurrent disease in lung cancer [152,153]. The cytokeratin marker M30 was increased in sera of breast cancer patients but failed to show prognostic value [154]. Circulating DNA fragments During apoptosis, chromatin is characteristically cleaved into mono- and oligonucleosomal fragments that are released into the circulation after disintegration of the cellular membrane [10,14,51,53]. In serum and plasma, circulating nucleic acids are conserved by their close association with histone protein from further digestion by endonucleases [155,156]. Elevated levels of circulating DNA fragments have been found in the blood of patients with most types of malignancy (Table 4) including lung, gastrointestinal, colorectal, breast, gynecological, renal, and nasopharyngeal cancers as well as lymphoma [157 171]. However, diagnostic value is limited because nonmalignant conditions associated with high cellular turnover such as those arising from severe bacterial infections also cause elevation of DNA fragments in the circulation [162,165]. Additional qualitative investigations detecting characteristic mutations, alterations of microsatellites, and epigenetic changes might increase the specificity for tumor DNA and improve the diagnostic power for cancer disease [172 175]. The amount of circulating DNA has prognostic significance in various cancers [160,176 178]. The same applies to some of the tumorspecific features of the circulating DNA [168,173,174]. Concerning the monitoring of therapy, patients responding to chemo- and radiotherapy showed declining values whereas those with insufficient response exhibited constantly high or even increasing values. These observations were valid for patients with various solid tumors as well as for lymphoma [157,159,164,165,167,169,179,180]. Several studies have focused on measuring the initial increase of circulating DNA fragments shortly after application of antitumor therapy [162,164,165,169]. A similar phenomenon has been described for EBV-DNA in the plasma of patients with nasopharyngeal cancer during radiotherapy [181]. The extent of these early changes of circulating DNA fragments in the serum of patients with lung cancer during chemotherapy is reported to predict the response to therapy [182].

they can contribute in assessing the extent and aggressiveness of disease. Additionally, many of them show prognostic or predictive information in various cancers. As they are easily and noninvasively accessible in blood, they offer the possibility for serial investigations. The temporal rise of circulating apoptotic markers during chemo- and radiotherapy shows high potential to objectively assess disease management and to predict treatment response in advance of current methods. Large-scale studies of the apoptotic markers evaluated in conjunction with multiple clinical parameters and other biochemical markers are encouraged to more fully establish the utility of the apoptotic markers in cancer management.

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