Disseminated tumour cells

Abstract Metastatic spread is a major factor in the prog-nosis of cancer patients. Early detection and eradicationof circulating tumour cells prior to the development ofmetastases could help to improve the outcome of patientsafter tumour resection. Disseminated tumour cells havebeen detected in different compartments of the body us-ing cytological and immunostaining methods and, morerecently, using different molecular biological techniques.The most frequently studied body compartments are thebone marrow, peritoneal cavity, blood and lymph nodes,but other body fluids such as urine, bile, pancreatic juiceand sputum have also been analysed. At all of these sites,tumour cells have been detected. However, the specificityand sensitivity of the methods and their prognostic impactare still being debated. This review discusses the accura-cy of the detection methods and the prognostic value ofdetecting disseminated tumour cells in the bone marrow,blood and peritoneal lavage of patients with colorectal,gastric and pancreatic carcinomas.

Keywords Disseminated tumour cells ·Immunocytochemical detection · Molecular detection ·Prognosis · Gastrointestinal carcinoma · Pancreaticcarcinoma

Introduction

Early tumour diagnosis, improved surgical treatment andmultimodal therapeutic concepts have helped to reduce themortality of patients with gastrointestinal and pancreaticcarcinoma. Nevertheless, the survival rates of these patientsare still highly unsatisfactory. The prognosis depends onlocal tumour recurrence and/or the occurrence of distantmetastases. Staging systems, such as the TNM classifica-

tion, have been developed to help estimate the probabilityof further tumour progression after treatment. Detection ofdisseminated tumour cells at a distance from the primarytumour before the development of metastases or local tu-mour recurrence may help to refine the TNM system [22].

In recent years, numerous immunocytochemical andmolecular studies have shown that micrometastases anddisseminated tumour cells can be demonstrated not onlyin lymph nodes that had appeared tumour free on conven-tional histology [37], but also in such body compartmentsas the bone marrow, peritoneal cavity and blood [15, 16,25, 54, 55, 59]. Not only have increasing numbers ofcompartments been analysed; studies have been extendedto other malignancies that seldom demonstrate metastaticspread to the bone, such as oesophageal, pancreas or pri-mary hepatic tumours [32]. From these data, it has beenconcluded that conventional staging most likely underes-timates the true tumour stage. However, because the tech-niques used to identify disseminated tumour cells havenot yet been standardised, the detection of disseminatedtumour cells was not included in the most recent TNMclassification. It does, however, recommend documentingany relevant findings. If isolated tumour cells are detect-ed immunocytochemically in lymph nodes, the UICCsuggests adding an “i+” in parentheses along with the Nstage. If molecular findings indicate the presence of tu-mour cells, a “mol+” should be added [23].

Disseminated tumour cells are detected by means ofimmunocytochemical or molecular methods designed torecognise certain tumour-associated antigens and/or celllineage-specific markers, such as cytokeratins (CKs) [15,16, 43, 54]. These markers are not tumour specific, how-ever, but may also be expressed by non-neoplastic cells.If both the immunocytochemical and the molecular ana-lyses are to be valid, it is therefore of the utmost impor-tance to avoid generating “false-positive” data, i.e. re-cognising non-neoplastic cells instead of tumour cells.However, a high degree of sensitivity must be reached toreduce “false-negative” results.

Another crucial issue is the biological significance ofthe disseminated tumour cells that are detected. Are these

I. Vogel (✉ ) · H. KalthoffMolecular Oncology Research Laboratory,Department of General and Thoracic Surgery, University of Kiel,Arnold-Heller-Strasse 7, 24105 Kiel, Germanye-mail: [email protected].: +49-431-5974481, Fax: +49-431-597-1939

Virchows Arch (2001) 439:109–117DOI 10.1007/s004280100476

R E V I E W A RT I C L E

Ilka Vogel · Holger Kalthoff

Disseminated tumour cellsTheir detection and significance for prognosis of gastrointestinaland pancreatic carcinomas

Received: 9 May 2001 / Accepted: 9 May 2001 / Published online: 7 July 2001© Springer-Verlag 2001

cells able to proliferate and give rise to metastases by in-teracting with the environment, or are they in a state ofdormancy, arrested in the G0 phase [58] and waiting toproliferate at a later point in time? An important answerto these questions will probably come from studies onthe prognostic relevance of disseminated tumour cellsdetected in the bone marrow, peritoneal cavity and blood.This article reviews these issues by discussing the vari-ous detection methods used so far and the prognostic rel-evance of the results gained from these studies.

Detection methods

Sensitivity and specificity

The detection rate of disseminated tumour cells is affected by vari-ous factors, including number of analysed cells, collection andtreatment of the sample, cell separation protocol, selection of anti-bodies and primers, and evaluation techniques.

The number of cells investigated clearly determines the detec-tion rate in a given patient, although the sensitivity of the variousassays is high, ranging from one tumour cell in 106 to one in 107

mononuclear cells [27, 52, 66]. It has also been demonstrated thatmultiple samples taken from different sites result in higher detec-tion rates than single samples [53].

All methods rely on the recognition of antigens or gene tran-scripts that are expressed only by tumour cells and not by the sur-rounding cells. It is often difficult to find such a specific antigen,but, even if there is a marker supposed to be expressed by the tu-mour cells, antigen expression in the disseminated tumour cellscan vary greatly [33], because antigens expressed by the primarytumour may have been downregulated or lost. For some markers[prostate-specific antigen (PSA), mucins], modulation due to hor-monal influence has been demonstrated [28]. This problem mightbe overcome by the parallel use of multiple markers.

False-positive results can also occur regardless of the methodsapplied [immunostaining or reverse-transcription polymerase chainreaction (RT-PCR)], if epithelial markers are used, due to contami-nation with skin cells or release of epithelial cells in benign prolif-erative diseases [24, 52, 72]. The isolation of mononuclear cells[34] may lead to loss of tumour cells and result in false-negativedata due to small sample volumes.

Immunostaining approaches

Several studies have clearly demonstrated that immunocytochem-istry is superior to conventional cytology at detecting isolated tu-mour cells in the bone marrow [42, 61]. Immunostaining methodsfor detecting disseminated epithelial tumour cells in mesenchymalcompartments, such as the bone marrow, employ antibodies direct-ed against a variety of epithelial cell markers. Pan-specific anti-bodies against CK seem to have a higher specificity than antibod-ies directed against the epithelial mucin family [14, 61] (Fig. 1).However, in addition to increased sensitivity, a higher number offalse-positive results due to non-specific labelling have been ob-served. The main reasons were (i) expression of some epithelialantigens by a few haematological cells [5], (ii) endogenous alka-line phosphatase in cases where anti-alkaline phosphatase (AP-AAP) complexes were used [3] and (iii) phagocytosed stainablematerial in granulocytes [48].

Molecular biological approaches

Chromosomal abnormalities can be detected in haematologicalmalignancies using breakpoint-specific PCR tests. In solid tu-mours, this molecular approach is not applicable, because the chro-

mosomal abnormalities are much more heterogeneous and com-plex. However, in solid tumours, it is possible to identify dissemi-nated tumour cells by detecting RNA specific to the tissue of ori-gin of these cells [6]. The method of choice is the RT-PCR, whichhas been further refined by many investigators so that it is applica-ble to the detection of disseminated tumour cells.

When disseminated tumour cells are analysed using RNA-based assays for epithelial cells, it has to be assumed that cells ofnon-haematopoietic origin normally do not circulate in the pe-ripheral blood or bone marrow. The target RNA should not be ex-pressed by haematopoietic cells, but exclusively by the dissemi-nated tumour cells that are to be identified. Alternatively, a two-step procedure of enrichment and/or derichment methods needs tobe included. Studies of target RNAs for tumours of gastrointesti-nal origin have mainly focused on tissue-specific markers, suchas CKs (CK19, CK20; Fig. 2) and mucins, or on tumour-associat-ed antigens such as carcinoembryonic antigen (CEA) [15, 16, 25,56, 59]. The PCR assays can detect a single target cell in up to100 million irrelevant cells in spiking experiments [10, 66]. Di-rect comparison of molecular methods using immunostaining ap-proaches showed that the PCR method is potentially 100 timesmore sensitive [64]. Moreover, by means of the PCR technique,target nucleic acids can be amplified by a factor of 106–109 with-in less than 1 h. The high sensitivity and specificity of these as-says can be limited, however, by DNA contamination [35], “ille-gitimate” expression (transcription of any gene in any cell type)of epithelial or tumour-specific antigens in haematopoietic cells[5, 11, 29], upregulation of target mRNA expression by inflam-mation or hormonal induction of gene expression [28], the exis-tence of pseudogenes, which can lead to false-positive results inhealthy individuals [28, 29, 47] or in patients with benign diseas-es [16], or the degradation of RNA during the various steps of theassay protocol [31].

The detection rates achieved for venous blood samples usingRT-PCR are lower than corresponding analyses in bone marrowsamples. Possible explanations for this phenomenon are (i) quali-tative and quantitative differences between cells in the bone mar-row and venous blood, (ii) differences in survival or immune es-cape possibilities in different compartments and (iii) methodolog-ical aspects, such as loss of tumour cells due to masking withthrombocytes or clotting problems.

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Fig. 1 Disseminated tumour cell in the bone marrow of a patientwith colorectal carcinoma. Using the alkaline phosphatase withanti-alkaline phosphatase (APAAP) technique, staining was per-formed with a cocktail of the following antibodies: C1-P83 [carci-noembryonic (CEA)], Ca 19–9, KL-1 (cytokeratin); counterstain-ing with haemalum

Characterisation of disseminated tumour cells

In addition to detecting disseminated tumour cells, it is necessaryto characterise them in more detail, especially because of thera-peutic implications. Features that are of interest in this respect arethe immunogenic potential of the cells, their proliferation activity,and their expression of growth factors and adhesion molecules.The identification of these features on disseminated tumour cellsrequires double labelling of the cells, which has been performedusing various techniques. Studies using these methods revealeddownregulation of major histocompatibility complex (MHC)class-I antigen in colorectal and gastric carcinomas [51, 62], neo-expression of intercellular adhesion molecule (ICAM)-1 and mel-anoma cell adhesion molecule (MUC-18) and downregulation ofdesmosomal protein [53], reduced expression of Ki67 (prolifera-tion-associated antigen) in gastrointestinal carcinoma [51], re-duced expression of PCNA (proliferating cell nuclear antigen) inprostatic cancer [45], expression of growth factor receptors(transferrin, c-erbB2) in metastatic breast cancer [51], expressionof E-cadherin (adhesion molecule) in breast and gastric cancer[13] and expression of the urokinase plasminogen activator (uPA)receptor [1].

New methods have been developed in recent years by combin-ing either immunostaining methods or immunofluorescence within situ hybridisation (FISH) [30, 39, 44] or single-cell PCR tech-niques [33]. With these techniques, it was possible to prove themalignancy of cells by detecting identical chromosomal abnormal-ities to those in the primary tumour [45]. These techniques havealso been used to demonstrate the heterogeneity of disseminatedtumour cells found at different sites in a single patient [33]. Cul-tures of disseminated cells have been successfully established invitro, increasing the cell number available for further phenotypicand genotypic analysis [49, 58].

Results of the clinical studies and prognostic impact

Colorectal carcinoma

Immunocytochemical analyses of bone marrow samplesfrom patients with colorectal carcinoma were initiallyperformed by Schlimok et al. [61] with a monoclonal an-tibody called CK2, which specifically reacts with CK18

[9]. Further studies with the same antibody revealed de-tection rates in the bone marrow between 16% and 32%.Other studies using combinations of antibodies founddisseminated cells in the bone marrow in up to 74% ofthe cases [4], whereas PCR tests yielded positive resultsin 31–84% of the patients [26]. Specificity was evaluatedin all studies by analysing samples from patients withoutany evidence of carcinoma or from healthy subjects.Most of the studies had a false-positive rate of below10% [27, 52, 65, 78]. Using multivariate analysis, Linde-mann [38] showed that detection of disseminated tumourcells in the bone marrow with the monoclonal antibodyCK2 is an independent prognostic factor for survival(Table 1). Leinung et al. [36] used the pan-specific CKantibody A45-B/B3, which detects a common epitope ona variety of CK types, including CK8, CK18 and CK19[68], and also found that the detection of disseminatedtumour cells had a significant influence on survival. Oth-er authors combined different antibodies directed againstCKs and/or tumour-associated antigens, but none ofthem performed multivariate analyses to demonstrateprognostic relevance [4, 27, 65]. To date, none of thestudies analysing peritoneal washings has demonstratedthat the detection of disseminated tumour cells in pa-tients with colorectal carcinoma is an independent prog-nostic marker [4, 27, 65] (Table 1).

Most molecular biological studies have been per-formed on bone marrow and/or venous blood from pa-tients with colorectal carcinoma. CK genes, especiallythe CK19 and CK20 genes, have been used as markergenes [7, 10, 12, 17, 59, 66, 75, 77, 78]. The gene of theCEA was also tested in several studies [26, 59, 69].Since haematopoietic cells can “illegitimately” expresstumour-associated antigens or epithelial cell-specific an-tigens [17, 79] and pseudogenes may give rise to PCRproducts identical to those of marker genes, the RT-PCRassays resulted in a large number of false-positive sig-nals [17]. So far, the CK20 gene seems to be the bestmarker, although some false-positive results have beenobserved with this also [75, 76]. Our analyses of 295 pa-tients with curative resections showed for the first timethat the detection of disseminated tumour cells in thebone marrow and/or venous blood is an independentprognostic factor for overall survival in patients with co-lorectal carcinoma [75] (Table 2). It was clearly demon-strated that tumour cells are present in the bone marrowand blood in colorectal carcinoma patients. Detection

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Fig. 2 Cytokeratin 20 (CK20)-specific nested reverse-transcrip-tion polymerase chain reaction (RT-PCR) after serial dilution ofRNA from the pancreatic tumour cell line A818–4 (cells per milli-litre). Separation of samples was done by agarose-gel electropho-resis and staining with ethidium bromide. The corresponding con-trol reaction was performed as GAPDH RT-PCR (359-bp product)using a duplex approach. ex RT-PCR product (788 bp) using theexternal primers, in nested PCR product (485 bp), –RT amplifica-tion without reverse transcription, K water control, M molecularweight marker

rates were higher in patients whose tumours were in anadvanced stage than in those with early tumour stages,although disseminated tumour cells were already detect-able in up to 20% of patients with early tumour stages[75]. Only a few other studies have also focused on the

prognostic relevance of tumour cell detection (Table 2).They confirmed that disseminated cells are of prognosticvalue for these patients, but the results need to be estab-lished by multicenter clinical trials under standardisedconditions.

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Table 1 Prognostic relevance of immunocytochemical studies on bone marrow (BM) and/or peritoneal lavage (PL) obtained from pa-tients with colorectal carcinoma prior to tumour resection

+: relevant to prognosis, –: not relevant to prognosis, ∅ : not done; univar.: univariate analysis; multivar.: multivariate analysis

Table 2 Prognostic relevance of molecular biological studies (PCR) on bone marrow (BM) and/or peripheral blood (BL) obtained frompatients with colorectal carcinoma prior to tumour resection


Gastric carcinoma

In immunocytological studies of patients with gastriccarcinoma, the same monoclonal antibodies have gener-ally been used to detect disseminated tumour cells in thebone marrow and the peritoneal cavity as in colorectalcarcinoma patients. Detection rates in the bone marrowranged between 25% and 82% [1, 4, 27]. Overall, thisseems to be slightly higher than in colorectal carcinoma.Even higher detection rates (32–72%) were observed inthe peritoneal cavity [4, 27, 65]. The explanation for thismight be the higher rate of peritoneal carcinomatosis atthe time of the primary operation in gastric carcinomacompared with colorectal carcinoma.

One study, published by Jauch in 1996 [24], includeda multivariate analysis. It demonstrated that the presenceof three or more cells in the bone marrow was of prog-nostic significance for disease-free survival, but only inpatients with T1/2 tumours (UICC classification; Ta-ble 3). Heiss et al. [21] showed that tumour cell dissemi-nation in the bone marrow was an additional prognosticfactor in patients who are in early tumour stages (UICC

I/II), are lymph node-negative (N0) and express PAI-1(plasminogen activator inhibitor type 1).

Molecular analyses have been performed with CEA,CK19 and CK20 as markers [7, 25, 59, 67]. To date, theonly studies to demonstrate in univariate analyses a sig-nificant difference in overall survival for patients withtumour cells in the bone marrow and blood were doneby our group (Table 4). These studies included 22 bonemarrow samples and 58 venous blood samples [67]. It israther striking that the detection rate in the bone marrowwas low relative to immunocytochemical studies, al-though advanced stages were included [67]. The likelyexplanation for this is that CK20 is not expressed at theprotein level in every gastric carcinoma [43]. It is there-fore necessary to evaluate other markers that might bemore consistently expressed in gastric carcinomas.

Pancreatic carcinoma

Still fewer studies have dealt with patients with pancreat-ic ductal carcinoma. This may be a reflection of the poor

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Table 3 Prognostic relevance of immunocytochemical studies on bone marrow (BM) and/or peritoneal lavage (PL) obtained from pa-tients with gastric carcinoma prior to tumour resection


Table 4 Prognostic relevance of molecular biological studies (PCR) on bone marrow (BM) and/or peripheral blood (BL) obtained frompatients with gastric carcinoma prior to tumour resection


prognosis for these patients and the lower incidence ofthe disease than with colorectal carcinoma. Immunocyto-chemical analyses of bone marrow samples have beenperformed using cocktails of antibodies, mostly includ-ing Ca 19–9 as a tumour-associated antigen [60, 70, 71,74]. In view of the higher number of advanced-stage pa-tients in the respective series, detection rates of dissemi-nated cells of almost 40–60% have been found [60]. Inunivariate analyses, most of the studies demonstratedshorter survival in patients in whom tumour cells weredetected in the bone marrow, while our investigationswere the only ones to demonstrate that detection of cellsin the peritoneal lavage has an impact on patient progno-sis [74] (Table 5).

Molecular analyses have been performed in only asmall number of cases [2, 50, 57, 66, 67]. Neither detec-tion in the bone marrow nor detection in the blood hasbeen evaluated for prognostic impact. The only study sofar to test mutant- Ki-ras (that is known to occur fre-quently in ductal carcinomas but also in hyperplasticduct lesions of the pancreas [40]) as a marker in theblood was on a series of ten patients [50]. Bilchik et al.[2] reported a PCR combination of three markers: MET(hepatocyte growth factor receptor gene c-met), Gal-NAc-T (β1,4-N-acetyl-galactosaminyl-transferase) andβ-hCG (β-human chorionic gonadotropin) in an analysisof 33 patients. Their detection rate was 100% in stage-IV patients and 87.5% in stage-II and -III patients, if allthree markers were combined. Obviously, these markerswill need to be evaluated in a larger series.

Conclusion and perspectives

Our evaluation of the studies on colorectal, gastric andpancreatic ductal carcinomas indicates that detection ofdisseminated tumour cells in different compartments maylead to more accurate tumour staging. Further studies will

be required, however, to verify the independent prognos-tic impact in each of the compartments analysed so far.Such studies should try to evaluate methodological as-pects, such as methods for enriching mononuclear or tu-mour cells, and develop highly specific and sensitive de-tection systems (including methods, marker and primerchoices). Before these methods can be incorporated intoclinical routine, they need to be standardised.

Further studies should focus, in particular, on (semi)-quantitative RT-PCR, which additionally allows the am-plification rate to be standardised. PCR reactions usingmultiple markers may overcome tumour cell heterogene-ity and false-positive results and would therefore increasethe sensitivity and specificity. The enrichment of tumourcells, e.g. by magnetic beads, could solve the problem offalse-positive results by reducing the background noise.Initial analyses using this method have been very promis-ing [10, 46].

If future studies confirm the results published so far,patients with proven disseminated tumour cells whohave no other clinical or radiological evidence of diseaseafter resection of the primary tumour should receive ad-juvant therapy, because conventional staging underesti-mates the lymphatic and haematogenous spread of thetumour. As recurrences are sometimes seen after a periodof years, it is likely that disseminated cells can persist ina dormant state for prolonged periods. There is some evi-dence that these cells cannot be reached by means ofconventional chemotherapy but might be better targetedby antibody-based adjuvant cancer therapies [41, 73].

One problem that has to be considered is the heteroge-neity of solid tumours, since it reduces the likelihood thatall disseminated cells will be removed. An individualisedcharacterisation of the tumour cells might be one solu-tion, but this does not appear practicable in daily routine.The use of a cocktail of antibodies is more promising. Fur-ther analyses that consider methodological aspects, cellcharacterisation, the prognostic impact and new adjuvant

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Table 5 Prognostic relevance of immunocytochemical studies on bone marrow (BM) and/or peritoneal lavage (PL) obtained from pa-tients with ductal pancreatic carcinoma prior to tumour resection


therapeutic approaches are necessary and will certainlylead to improvements in the treatment of gastrointestinaland pancreatic carcinoma in the near future.

Acknowledgements The authors thank Prof. Klöppel for criticalreading the manuscript. They are also thankful to Mrs. Kay Degeand Heinke Brütting for English editing and typing the manu-script.

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