Abstract
Intracellular macromolecules are released from dying tumor cells and may subsequently be detected in patient blood. In this review, we will discuss the use of cytokeratin-18 as a serum biomarker for monitoring therapy-induced cell death. Cytokeratins are abundant intracellular proteins expressed by most types of carcinoma, but not by treatment sensitive cells from bone marrow and other tissues. Release of cytokeratins into blood is therefore expected to show some specificity for tumor cell death. Cytokeratin-18 (CK18) is cleaved by caspases specifically during apoptosis, and the molecular form of this protein (caspase-cleaved vs. non-cleaved) released from dying tumor cells is therefore diagnostic as to the type of cell death (apoptosis vs. necrosis). Analyses of different CK18 forms in patient sera have suggested that tumor apoptosis may not necessarily be the dominating death mode in many tumors in vivo. Measurements of increased levels of CK18 in serum during therapy of prostate and breast cancer patients have been encouraging with regard to the possible future use of CK18 as a biomarker for monitoring therapy efficiency.

1. Introduction
The possibility to predict which patients that will respond to a particular treatment modality is becoming increasingly important. With a widening arsenal of cancer therapies available, the clinician must receive guidance as to which patients should be treated with which drug. Ideally, biological markers will be available for predicting whether a specific tumor will be sensitive to treatment. For some types of therapies, such markers are available. Estrogen receptor status is an important parameter in predicting the likelihood of response of breast carcinomas to tamoxifen. As a first line therapy, tamoxifen induces a response in up to 80% of all estrogen receptor positive tumors, but only in 10% of estrogen receptor negative tumors. Treatment with Herceptin, a humanised HER2 monoclonal antibody, results in prolonged survival of patients with HER2 overexpressing breast cancer cells, but is ineffective in other breast cancer patients. Unfortunately, many years of research has failed to identify reliable markers for prediction of sensitivity to chemo- or radiation therapy, probably because sensitivity to treatment by most agents is controlled by a large number of genes. Assessing the expression of one gene product - such as the P-glycoprotein - is therefore unlikely to be meaningful. Attempts to use microarray technologies to correlate the expression pattern of many genes with treatment sensitivity may turn out to be more successful. Whether "pharmacogenomics" will live up to current expectations as predictive tools is as yet unclear.

An alternative approach to treatment monitoring is to develop methods for assessment of cell death in real time. This approach is not truly "predictive", but offers potential advantages in terms of reliability and the possibility to follow treatment effects. Cell death monitoring can be achieved using imaging techniques such as ^99mTc-Annexin V scintigraphy [1] or through FDG-PET [2]. An attractive alternative, due to the ease and low cost, is to measure the levels of macromolecules released from tumor cells during therapy. The purpose of the current review is to discuss the assessment of acute cell death in patient serum samples.

2. Release of intracellular macromolecules from tumor cells into serum
It is well established that DNA levels are elevated in plasma from cancer patients [3, 4]. The origin of this plasma DNA has been debated, and argued to be derived from tumor apoptosis [4, 5] or from secretion of DNA by cells [6]. Chromatin is cleaved into oligo- and mononucleosomes during apoptosis by caspase-activated DNAase (CAD, [7]). The nucleosomes are subsequently destructed, leading to release of the core histones H2A, H2B, H3, and H4 from DNA [8]. Not all DNA released from apoptotic cells is free from histones since histone-DNA complexes ("nucleosomes") can be detected in plasma [9, 10].

With the introduction of polymerase chain reaction (PCR) methods, interest in analysing DNA in serum has increased and studies have been performed to specifically detect tumor DNA by analysis of tumor specific mutations, DNA methylation patterns and microsatellite instability [11-14]. Such measurements may be of clinical utility. Plasma DNA can be detected during early lung cancer recurrencies [15] and increased serum DNA levels correlate to overall survival in breast cancer [16]. Increases in nucleosome levels have also been documented to occur during therapy [17].

Since the original description of carcinoembryonic antigen (CEA) [18], a large number of cellular proteins have been shown to be elevated in sera from cancer patients. These proteins are generally referred to as "serum tumor markers" and can be used to monitor disease progression. Clinically useful markers include CEA, PSA, CA125, TPA/TPS, CA19-9 and CA15-3. The value of serum tumor markers for detection of early remission is reduced in clinical situations where no effective treatment can be offered. The present review does not aim to discuss the use of serum tumor markers; for reviews of this field please see [19, 20].

A more general approach to examining tumor markers is to study the patterns of tumor derived peptides in patient blood, "serum proteomics" [21]. It is hoped that cancer-specific peptide profiles can be detected using mass spectrometry and then used for diagnostic and prognostic purposes. This approach is complicated by the fact that the abundance levels of serum proteins vary tremendously (over a range of 10⁹). Tumor derived proteins are likely to be present in the low end of this spectrum (at pg/ml) and therefore difficult to measure against a background of albumin, haptoglobin, immunoglobulin and other abundant proteins (present at µg to mg/ml). An additional complication is that tumor cell proteins are usually not degraded to peptides in the tumor interstitial fluid [22], making detection by mass spectrometry difficult.

3. Release of macromolecules during cell death - apoptosis and necrosis
By what mechanism(s) are biomarkers released from tumor cells? Whereas some proteins such as alpha-fetoprotein and PSA are secreted by cells, it is clear that cell death is a major mechanism for the release of cellular macromolecules. Release of DNA from dying cells has been documented to occur in tissue culture [14, 23]. Similarly, intracellular proteins may be released by dying cells and detected in serum. Examples of such proteins are S100B [24], CA 125 [25] and cytokeratins [26-28] (see below).

Two major types of cell death are recognized: apoptosis and necrosis. Apoptosis is a regulated process that can be triggered by different stimuli and is mediated by a cascade of enzymes. Necrosis is a catastrophic form of cell death which does not involve the regulated action of enzymes. These two death modes are believed to be extreme forms observed in vitro, and the situation in vivo may be less clear cut [29]. Factors such as intracellular levels of ATP may decide whether an apoptotic stimulus induces apoptosis, or if the process shifts into necrosis [30]. Furthermore, apoptotic cell death pathways have been described which do not involve caspases or involve combinations of caspases and other effectors [31]. The mode of cell death in tumors, apoptosis, necrosis or intermediate forms, is likely to show large variations dependent on the genotype of the tumor cells and the tumor microenvironment.

How do intracellular proteins released by dying cells reach the circulation? Apoptosis results in fragmentation of cells into apoptotic bodies which are engulfed by neighbouring cells and macrophages. In situations of enhanced cellular turnover and cell death, such as in cancer, the local clearance mechanisms may be overloaded. Apoptotic bodies that are not engulfed by macrophages will disintegrate ("secondary necrosis") [32] and their contents may subsequently reach the circulation (Fig. 1A). Necrotic cells are also recognized by phagocytic cells by a phosphatidylserine-dependent mechanism [33]. However, uptake of necrotic cells has been reported to be less efficient than phagocytosis of apoptotic cells [33]. Whether differences in local clearance of apoptotic and necrotic cells will lead to a more efficient release of biomarkers from necrotic cells is not known, but this scenario is not unlikely.

Fig. 1. Theoretical scenarios of release of intracellular macromolecules from tumor cells. (A). Tumor cells die by apoptosis and necrosis in the tissue. Macromolecules released by the cells subsequently enter into the circulation. (B) Tumor cells that form parts of the vessel wall in mosaic vessels are shed into the circulation where they undergo apoptosis.

Tumor cells that form part of the vessel wall, forming so called "mosaic vessels", are shed into the circulation [34] (Fig. 1B). It has been estimated that approximately one million tumor cells are shed per gram of tumor per day [35, 36]. Circulating tumor cells have a high apoptotic index [36], and cells that die in the circulation will release intracellular proteins (Fig. 1B). It is not known to what extent "serum tumor markers" originate from tissue-bound cells that die and leak out material, compared to cells that are shed from tumors and subsequently die in the circulation.

The clearance rates of biomarkers in serum are important determinants of the steady-state concentrations. Free PSA has a half-life of approximately one hour [37] and appears to be removed from the circulation via the liver and kidneys [38]. S100B is cleared with a half-life of about 30 minutes [24]. Cancer patients may have impaired kidney functions leading to increased filtration of biomarkers into urine. In addition, anticancer drugs induce nephrotoxicity [39]. Apoptotic bodies containing caspase-cleaved cytokeratin-18 accumulate in the spleen [40]. Interestingly, approximately 80% of patients with metastasized cancer showed accumulation of apoptotic bodies in the red pulp of the spleen [40].

4. Cytokeratins as serum tumor markers
Cytokeratins are members of the family of intermediate filament proteins and are found primarily in epithelial cells [41]. Type I cytokeratins are acidic and type II are neutral to basic proteins. Cytokeratin filaments are heteropolymers between type I and type II proteins. Cytokeratins 8, 18 and 19 are expressed by most types of carcinomas, including those of the breast, prostate, lung, colon and ovary.

Cytokeratins are released from tumor cells and provide useful serum markers for evaluating the clinical progression of patients with epithelial malignancies [42, 43]. Tissue polypeptide antigen (TPA) was originally identified as a tumor antigen present in the insoluble fraction of human tumor cells [44], and was later shown to consist of fragments of cytokeratins 8, 18, and 19 [45]. Tissue-polypeptide-specific antigen (TPS) is defined by a monoclonal antibody recognizing a C-terminal epitope on cytokeratin 18 [46], and CYFRA 21-1 detects cytokeratin 19 [47].

Cytokeratins can be demonstrated in tumor necrotic areas by injection of labelled antibodies [48]. Cytokeratins are also released into the circulation where they may comprise partly degraded intermediate filament complexes [42]. It is also known that proliferating cells have a substantial pool of soluble cytokeratin 8 and 18 [49], which is released after loss of membrane integrity [28]. Soluble cytokeratin fragments can also be produced by caspase-cleavage during apoptosis (Fig. 2), and these fragments are relatively stable [50, 51].

Fig. 2. Cleavage of cytokeratin-18 by caspases. CK18 is cleaved at two positions by caspases. Cleavage at the C-terminus leads to formation of a neo-epitope recognized by the monoclonal antibody M30.

The cytokeratin tumor marker field has recently been developed by the introduction of a monoclonal antibody which recognises a neo-epitope of CK18 exposed after caspase cleavage during apoptosis [52]. The antibody, M30, detects only caspase-cleaved fragments of CK18, but not the native protein (Fig. 2). An ELISA assay based on the M30 antibody has been developed and made commercially available [53]. Elevated levels of caspase-cleaved CK18 in serum were associated with an ER-negative status of tumour tissues [54], consistent with reports of ER-negative tumors having higher apoptotic indeces [55]. In patients with recurrent breast cancer, serum levels of caspase-cleaved CK18 correlated with the number of involved organs and performance status [54].

Fig. 3. Release of different molecular forms of CK18 during apoptosis and necrosis.

Measuring the ratio of caspase-cleaved to total CK18 released from cells is a convenient method to assess cell death mode [28] (Fig. 3). ELISA assays for determining these different molecular forms that use the same recombinant standard are available for such determinations (PEVIVA AB, Bromma, Sweden). We have observed that in venous blood collected from endometrial tumors during operation, only a fraction of the total CK18 reacts with the M30 antibody. This suggests that serum CK18 is not generated by "pure" apoptosis in most instances [28], and that necrosis is a dominating death mode in vivo. Previous studies have used the size of DNA in patient plasma as an indicator of tumor death mode, where the presence of smaller DNA fragments are believed to reflect release of nucleosomes from apoptotic cells and higher molecular weight DNA molecules believed to reflect release from necrotic cells [14, 56, 57]. It has recently been demonstrated that injection of necrotic cells into mice leads to the appearance of low molecular weight DNA, probably derived from macrophages that undergo overload-induced apoptosis [10]. Macrophages do not express CK18 and it does not seem likely that tumor cell derived CK18 will become caspase-cleaved after phagocytosis by macrophages. CK18 measuments are therefore expected to be more accurate for determination of cell death modes compared to measurements of DNA size.

Studies of serum DNA have shown that tumor DNA may constitute as little as 3% of total ciculating DNA [14]. Which exact cell types that release the major part of the DNA found in the blood is not clear; dying T-cells were only occasionally observed to do so. Macrophage apoptosis due to overloading of their phagocytic capacity is another possibility [10]. It is also possible that tumor expansion may lead to the death of normal cells in the surrounding tissue. Finally, tumors may secrete cytokines such as FasL or TNF-alpha that may induce cell death in other organs. We have found that the levels of CK18 are higher in local tumor veins compared to peripheral blood in the same patients [28], strongly suggesting that CK18 is released by the tumor or the immediately adjacent tissue.

5. Using serum cytokeratins to measure acute cell death
Since serum tumor markers often are release by dying cells, they could potentially be used to assess cell death induced by cancer treatment. However, the understanding that various serum markers are produced by cell death is fairly recent, and their potential in assessment of cancer treatment-induced cell death has not been widely exploited.

Cytokeratin 18 has three major advantages as a biomarker for tumor cell death:

(i) the epithelial specificity of cytokeratins restricts the possible cell types that can release this biomarker. None of the cell types that normally circulate in blood express CK18, neither do dividing and chemotherapy-sensitive cells of the bone marrow.

(ii) cytokeratins are abundantly expressed. Cytokeratins are major intracellular proteins, readily visible on one-dimensional SDS-PAGE gels of total extracts of epithelial cells. This high level of expression is of course advantageous for subsequent tracing in serum. Theoretical calculations suggest that apoptosis of as little as 10-20 million carcinoma cells can be detected in the serum of a cancer patient: one dying cell generates 10^-5 U of caspase-cleaved CK18 measured by the M30-Apoptosense assay and therefore 200 U in 3 L of plasma. Increases of < 50 U/L in plasma are readily detected by the M30-Apoptosense ELISA assay.

(iii) cytokeratin 18 is cleaved by caspases during apoptosis. As described above, the molecular form of CK18 released from cells can be used to determine death mode - apoptosis or necrosis.

We have observed increased levels of caspase-cleaved CK18 in serum during therapy [28, 58]. These increases were primarily observed in patients that responded to therapy. Interestingly, in some responding patients increases in total CK18, but not in apoptosis-specific caspase-cleaved CK18, were observed [28]. Apoptosis may therefore not be the dominating death mode of the tumors of these patients. This finding is important with regard the interpretation of studies that have used apoptosis in tumor biopsies as a biological endpoint to examine therapy efficiency [59-61]. We do not believe that assessment of only apoptosis will be sufficient to allow prediction of therapy efficiency.

It is not known whether normal epithelial cells release proteins into the blood when they die, but it seems unlikely since increases in serum CK18 are not always observed during treatment of cancer patients with cytotoxic drugs. This heterogeneity in treatment response would not be expected if the drugs were to induce apoptosis of normal epithelial cells, leading to release of CK18 into the circulation. We would like to hypothesize that dying epithelial cells in normal tissue will shed/release their CK18 content into the lumen of the epithelia rather than into the circulation over the basal membranes. In contrast, the disorganized state of tumor tissue will lead to release into blood.

Measurements of CK18 release may be particularly useful in clinical situations which are difficult to evaluate with existing technologies. Many patients with advanced disease do not respond to therapy with radiologically measurable decreases in tumor mass, but rather with a more vague state of "stable disease". This category is likely to be quite heterogeneous, where some but not other patients benefit from chemotherapy [62].

Improved patient survival is the gold standard for approval of new drugs. However, the use of survival as a clinical endpoint requires long observation periods. Furthermore, the clinical efficiencies of investigational drugs are almost always assessed in patients with advanced disease, and almost invariably with disappointing results. The possibility to follow treatment effects in real time allows the clinician to adjust both the type of drug and the dose of the drug. This is a major advantage compared to studies of predictive markers. It would be very attractive to combine CK18 measurements with measurements of other biomarkers released by sensitive normal cells to be able to determine the therapeutic window. We are planning such studies.

6. Conclusions
Any method for treatment monitoring to be introduced into routine clinical care has to be reasonably simple and affordable. Assessment of tumor cell death in blood as a marker for therapy response meets these basic criteria. Using this method we have shown that the same treatment protocol may induce apoptosis in some tumors and necrosis in others. Clearly, both types of cell death must be assessed when monitoring therapy response. Although measurements of tumor cell death in blood is a promising therapy response marker, many questions remain to be answered with regard to the use of CK18 as marker of cell death, including which cells (tumor or normal) that release the macromolecule, the mechanisms of release of CK18 into circulation and the mechanisms of clearance.

Acknowledgements
The authors would like to thank Cancerföreningen in Stockholm for economic support for this project and thank Peter Björklund, PEVIVA AB, for comments.

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