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Detection and prognostic impact of disseminated tumor cells in pancreatic carcinoma
Review Pancreatology 2002;2:79–88
Detection and Prognostic Impact of Disseminated Tumor Cells in Pancreatic Carcinoma Ilka Vogel Holger Kalthoff Doris Henne-Bruns Bernd Kremer Molecular Oncology Research Laboratory, Department for General and Thoracic Surgery, University of Kiel, Germany
Key Words Disseminated tumor cells W Minimal residual disease W Pancreatic carcinoma
this increased staging has an prognostic impact and can be useful for therapeutic decisions in patients with pancreatic carcinoma. Copyright © 2002 S. Karger AG, Basel and IAP
Abstract Background/Aims: Metastatic disease determines the prognosis of patients with pancreatic cancer. Current routine staging methods often underestimate the tumor stage because they do not include the search for disseminated tumor cells that spread early in different compartments of the body. Immunohistochemical and molecular methods developed recently are able to detect these cells in multiple compartments of the body. Methods: The current status of the detection and the prognostic impact of disseminated tumor cells detected in lymph nodes, bone marrow, blood and peritoneal lavage of patients with pancreatic carcinoma are reviewed. Results: Disseminated tumor cells can be detected in different compartments of the body even in early tumor stages and when a resection of the primary tumor in curative intention was performed. Furthermore, the detection of these cells has importance for the prognosis and therefore will have therapeutic implications. Standardization of the methods is a prerequisite for further studies. Conclusion: The detection of disseminated tumor cells should be included into studies to reveal that
ABC
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Introduction
The prognosis for patients with ductal pancreatic adenocarcinoma remains poor. Although operation techniques, multimodal treatment and lower postoperative mortality could improve survival, overall 5-year survival rates range between 5 and 20% [1, 2]. Only 10–20% of the patients diagnosed with pancreatic head carcinoma are resected with a curative intention [3]. But even in these patients tumor recurrence is frequent because of early lymphatic and haematogeneous distribution and consecutive development of distant metastases, local recurrence and peritoneal seeding. Assessment of lymph node status as an indicator for tumor dissemination is routinely done by histopathological examination. Tumor-associated antigens and/or tissue specific markers have been used to detect disseminated tumor cells in immunohistochemical analyses and have also improved the results of detection by conventional cytology [4]. Molecular biological approaches have been developed in recent years and focus mainly on
PD Dr. med. Ilka Vogel Klinik für Allgemeine Chirurgie und Thoraxchirurgie Forschungsgruppe Molekulare Onkologie, Universitätsklinikum Kiel Arnold-Heller-Strasse 7, D–24105 Kiel Tel. +49 431 597 4481, Fax +49 431 597 1939, E-Mail [email protected]
tumor-specific antigens and tissue-specific markers [5–8]. Additionally, bone marrow, blood and peritoneal washings have been analyzed (see tables 2–4). The studies demonstrate, that tumor staging can be improved by these methods but a definitive assessment of whether these cells are of prognostic relevance is complicated by the fact that many different methods and markers have been used with multiple detection systems. This review describes and summarizes the results of those studies, which analyzed disseminated tumor cells in lymph nodes, bone marrow, venous blood, peritoneal washings, and other compartments in patients with pancreatic carcinoma.
Results
Detection Methods – Methodical Aspects The detection of disseminated tumor cells depends on a number of steps including collection and treatment of the sample, cell separation protocol, chosen antibodies, number of analysed cells and evaluation techniques. All methods used depend on the recognition of antigens or gene transcripts that are believed to be exclusively expressed by tumor cells and not by surrounding cells in the examined compartment. In all methods (immunostaining or RT-PCR) unwanted positive results could occur due to contamination with skin cells, release of epithelial cells in benign proliferative diseases if epithelial markers are used [9, 10] or in case of an ectopic expression of epithelial markers in mesenchymal cells; false negative results may occur due to losses of tumor cells during isolation of mononuclear cells [11]. Immunostaining Approaches Immunostaining methods to detect epithelia-derived disseminated tumor cells in mesenchymal compartments like bone marrow exploit monoclonal antibodies directed against a variety of epithelia-specific cytoskeleton and membrane antigens (compare table 1–4). Several studies have clearly demonstrated that immunocytochemistry is superior to conventional cytology for the detection of isolated tumor cells in bone marrow [12] and peritoneal washings [13]. Pan-specific antibodies against cytokeratin seem to have a higher specificity than antibodies directed against the epithelial mucin family [14, 15]. Immunostaining lead to a further increase in sensitivity but a higher number of false positive results due to non-
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specific labeling were occasionally observed as well. The main reasons are: (i) expression of some epithelial antigens by a few hematological cells [16]; (ii) endogeneous alkaline phosphatase if anti-alkaline phosphatase (APAAP) complexes were used [17], and (iii) phagocytosed material in granulocytes than can be stained by antibodies [18]. Further investigations of new antibodies or combinations of antibodies that are highly specific and sensitive in their detection system are necessary. Depending on the detection system different authors have found in cell spiking experiments that it is possible to detect one tumor cell in 106 cells on a cytospin by immunocytochemical methods [9, 13]. This rate can be further increased by enrichment methods such as immunomagnetic beads isolation protocols [19]. To incorporate these methods into a clinical routine, it is necessary to standardize the methods used to date. Work on standardization of the evaluation of disseminated cells by immunohistochemical methods is currently in progress [17]. Molecular Biological Approaches Molecularbiological detection methods can be divided into two groups: (1) Detection of tumorspecific chromosomal abnormalities or mutations: In solid tumors the chromosomal abnormalities are heterogeneous and complex. An analysis of chromosomal abnormalities is therefore mainly restricted to tumors with mismatch-repair gene deficiencies. The use of specific mutations such as in the K-ras or p53-gene as markers is possible but the feasibility is rather poor [20]. (2) Detection of marker-gene expression (m-RNA) by reverse transcriptase polymerase chain reaction: Another way is the detection of RNA specific to the tissue of origin by reverse transcriptase polymerase chain reaction (RTPCR). Because mRNA is unstable in the extracellular environment, its detection in tissue or fluid is strongly indicative for the presence of vital tumor cells. For analyses of disseminated tumor cells with RNA-based assays for epithelial cells it has to be assumed that cells of nonhematopoietic origin are normally not circulating in the peripheral blood or bone marrow. The target RNA should not be expressed by hematopoietic cells, but by the disseminated tumor cells to be identified. Therefore, cytokeratins, especially cytokeratin 20 (CK 20) have been used (tables 2, 3) as epithelial-specific markers and the tumor-associated antigens like CEA and Ca 19-9 in pancreatic carcinoma. The PCR assays can detect a single target cell in up to 100 million irrelevant
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Table 1. Detection of disseminated tumor cells in lymph nodes of patients with pancreatic carcinoma Marker/antibodies
Detection rates
Patients R-situation
n
R0
18
Prognostic relevance univariate analysis
Author
multivariate analysis
Immunocytochemistry Ber-EP4
16/37 (43.2%) N0 lymphatic nodes of 13/18 patients (72.2%)
+ (overall and relapse-free survival)
Hosch et al. [4], 1997
Molecular biology K-ras
R0 10 UICC-stage I–III
4/6 patients (66.6%)
+
n.d.
Tamagawa et al. [7], 1997
K-ras (PCR/RFLP)
R0 (stage I/II)
15
8/13 (61.5%) 42/101 lymph nodes, paraaortic
n.d.
n.d.
Ando et al. [8], 1997
K-ras (PCR/RFLP)
R0 (stage I)
22
16/22 (73%) one or more regional lymph nodes
n.d.
n.d.
Demeure et al. [5], 1998
K-ras (PCR/RFLP)
R0 (stage I)
25
17/25 (68%) one or more regional lymph nodes positive
– n.d. 6/17 pos. patients and adju. therapy: +
Demeure et al. [36], 1998
AE3/AE1 (IHC)
R0 (stage I/II)
30
IHC: 14/30 (46.7%) PCR: 19/30 (63.3%) combination 25/30 (83.3%)
–
Brown et al. [6], 2001
K-ras (PCR/RFLP)
n.d.
–
+ = Relevant to prognosis; – = not relevant to prognosis; n.d. = not done.
Table 2. Detection of disseminated tumor cells in bone marrow samples of patients with pancreatic carcinoma Marker/antibodies
Detection rates
Patients R-situation
n
CEA, Ca 19-9, 17-1-A, C54-0, Ra 96, KL-1
R0-R2
32
CK2, KL-1, A45-B/B3
R0-R2
CK2, KL-1, A45-B/B3
CK2, KL-1, A45-B/B3
Prognostic relevance
Author
univariate analysis
multivariate analysis
15/26 (58%)
n.d.
n.d.
Juhl et al. [13], 1994
42
24/42 (57%)
+ (R0)
n.d.
Thorban et al. [60, 61], 1996
R0-R2
48
14/31 (48%) resected patients 10/18 (59%) not-resected patients
+
n.d.
Roder et al. [62], 1999
R0-R2
48
25/48 (52%) 4 (8.3%) CK2 positive 16 (33.3%) KL1 9 (18.6%) A45- B/B3 positive +
n.d.
Thorban et al. [63], 1999
Immunocytochemistry
CEA, Ca 19-9, 17-1-A, C54-0, Ra 96, KL-1
R0-R2
80
27/71 (38%)
(trend)
n.d.
Vogel et al. [46], 1999
AE1/AE3
R0-R2 54 (UICC-stage I–V)
13/54 (24%) R0: 13%
–
–
Z’graggen et al. [42], 2001
CEA
no details
3
2/3 (66%)
n.d.
n.d.
Gerhard et al. [64], 1994
CK 20
R0-R2
11
4/11 (36%)
n.d.
n.d.
Soeth et al. [21], 1996
CK 20
R0-R2
27 (BM)
5/27 (19%)
n.d.
n.d.
Soeth et al. [38], 1997
Molecular biology
+ = Relevant to prognosis; – = not relevant to prognosis; n.d. = not done.
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Table 3. Detection of disseminated tumor cells in blood samples of patients with pancreatic carcinoma Marker/antibodies
Detection rates
Patients R-situation
n
R0-R2 (stage I–IV)
105
R0–R2
6
Prognostic relevance
Author
univariate analysis
multivariate analysis
27/105 (26%) 3/32 (9%) R0 patients
–
–
Z’graggen et al. [42], 2001
2/6 (33.3%)
n.d.
n.d.
Tada et al. [65], 1993
Immunocytochemistry AE1/AE3
Molecular biology K-ras CEA
R0-R2
9
3/9 (33%)
n.d.
n.d.
Funaki et al. [66], 1996
K-ras
R0-R2
10
0/10 intraoperative 50% detection rate
n.d.
n.d.
Nomoto et al. [67], 1996
CK 19
no details
49
2/49 (4%) venous blood 2/49 (4%) portal blood
n.d.
n.d.
Aihara et al. [40], 1997
CK 20
R0-R2
22
2/22 (9%)
n.d.
n.d.
Soeth et al. [38], 1997
CK 20
R2 (stage IV)
28
22/28 (78%)
n.d.
n.d.
Chausovsky et al. [68], 1999
Chymotrypsinogen
R0-R2 (stage I–IV)
10
7/10 (70%)
n.d.
n.d.
Kuroki et al. [69], 1999
CEA
R0-R2 (stage I–IV)
21
13/21 (61.9%)
n.d.
n.d.
Miyazono et al. [70], 1999
MET, GalNac-T, ß-hCG
R0-R2
33
17/17 (100%) stage IV n.d. 8/16 (MET) 8/16 (GalNac-T) 7/16 (ß-hCG) 2/16 negative for all 3 markers in stage II/III
n.d.
Bilchik et al. [41], 2000
CEA
R0-R2
27
11/27 (41%)
n.d.
Piva et al. [71], 2000
n.d.
+ = Relevant to prognosis; – = not relevant to prognosis; n.d. = not done.
cells in spiking experiments [19, 21]. Direct comparison of a molecular biological with immunostaining methods showed that the PCR test is potentially 100 times more sensitive [22]. The high sensitivity and specificity of these assays can be limited, however, by DNA contamination [23], ‘illegitimate expression’ of epithelial or tumor-specific antigens in hematopoietic cells [16, 24, 25], upregulation of target mRNA expression by inflammation or hormonal induction of gene expression [26], the existence of pseudogenes that can lead to false-positive results in healthy individuals [25–27] or in patients with benign diseases [28], or the degradation of RNA during the various steps of the assay protocol [29]. Further studies should focus on quantitative RT-PCR which allows standardization of the amplification rate [30]. PCR-reactions with multiple markers may overcome tumor cell heterogeneity and false positive results and
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may yield an increased sensitivity and specificity. The enrichment of tumor cells, e.g. by magnetic beads, could solve the problem of false positives by reducing the background. Initial analyses with this method have been very promising [19, 31].
Results of the Clinical Studies and Prognostic Impact Detection of Disseminated Tumor Cells in Lymph Nodes It is generally accepted that lymph node metastases and therefore the UICC tumor stages have a prognostic value in most solid carcinoma. Conventional routine clinical histopathologic examination of regional lymph nodes is generally performed on formalin-fixed and paraffin-embedded tissue sections after staining with hematoxylin and eosin (HE). Mostly,
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Table 4. Detection of disseminated tumor cells in the peritoneal cavity of patients with pancreatic carcinoma Marker/antibodies
Detection rates
Patients R-situation
n
R0-R2
20
R0-R2
40
R0-R2
Prognostic relevance
Author
univariate analysis
multivariate analysis
5/25 (25%)
n.d.
n.d.
Martin et al. [72], 1986
12/40 (30%)
+
n.d.
Warshaw et al. [43], 1991
Cytology
57
12/36 (33%)
+
n.d.
Heeckt et al. [73], 1992
R0-R2 36 (stage I–IV)
3/36 (8.3%)
(+)
n.d.
Lei et al. [74], 1994
R0
60
4/60 (7%)
–
n.d.
Leach et al. [75], 1995
R0-R2
94
16/94 (17%)
n.d.
n.d.
Fernandez-del Castillo et al. [76], 1995
R0-R2 228 (stage I–IV)
34/228 (15%)
+
–
Merchant et al. [44], 1999
CEA, Ca 19-9, 17-1-A, C54-0, Ra 96, KL-1
R0-R2
31
18/31 (58%)
n.d.
n.d.
Juhl et al. [13], 1994
Ca 19-9, CEA
R0-R2
20
4/20 (20%)
n.d.
n.d.
Nomoto et al. [39], 1997
Ca 19-9, B 72. 3, CEA Leu-M1 and cytology
R0-R2
137
32/137 (23%)
(+)
n.d.
Makary et al. [45], 1998
CEA, Ca 19-9
R0-R2
74
8/8 (100%) peritoneal carcinosis 14/66 (22%) without visible peritoneal carcinosis
–
n.d.
Nakao et al. [77], 1999
CEA, Ca 19-9, 17-1-A, C54-0, Ra 96, KL-1
R0-R2
80
24/62 (39%)
+
n.d.
Vogel et al. [46], 1999
K-ras PCR
R0-R2
24
2/24 (8%) Cytology 3/24 (12%)
n.d.
n.d.
Rall et al. [47], 1995
K-ras PCR
R0-R2
20
2/20 (10%)
n.d.
n.d.
Inoue et al. [48], 1995 Nomoto et al. [39], 1997
Immunocytochemistry
Molecular biology
+ = Relevant to prognosis; (+) = relevant to prognosis of a subgroup; – = not relevant to prognosis; n.d. = not done.
only some representative sections of the many lymph nodes are examined which leads to underestimation of the stage. Gusterson et al. [32] calculated, for breast cancer lymph nodes, that histopathologic examination has only a 1% chance to identify a small lesion smaller than 3 cells. The analysis of more sections increase the detection rate, but also do not reach the detection rates observed by immunocytochemistry. Hosch et al. [4] found by the use of the pancytokeratin antibody Ber-EP4 in 16 of 37 conventional histologically negative lymph nodes disseminated tumor cells by immunostaining. They could also demonstrate an influence on overall and relapse-free survival in multivariate analysis when negative and positive tested patients were compared.
Molecular assays based on the PCR further increased the detection rates (tables 1–4) [33, 34], but because the tumor cells are not histologically identified or visualized, there are some concerns especially from traditional pathologists whether the detection of a gene product indicates the presence of a tumor cell. As up to 95% of the pancreatic carcinoma harbor a Kras point mutation [35], the presence of the mutant gene in a lymph node was interpreted as a proof for metastatic ells in the lymph node examined. The first study from Tamagawa et al. [7] demonstrated positive findings in 4 of 6 patients, who were classified as N0 by conventional analyses K-ras mutations. Ando et al. [8] and Demeure et al. [5, 36] confirmed these results with detection rates
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from 61.5% up to 73% in histological negative lymph nodes. In a recent study by Brown et al. [6], the nodes were analyzed by stepwise serial section and immunocytochemistry for cytokeratin (antibody: AE3/AE1) and K-ras PCR in parallel. By immunocytochemistry 46.7% of the conventional negative lymph nodes showed disseminated tumor cells and in 63.3% of the cases a K-ras analysis was positive. If both analyses were combined there was evidence for tumor cells in 83.3% of the conventionally negative nodes. However, this group found no difference in survival between the group with or without detected tumor cells. Patients with disseminated tumor cells who received adjuvant chemoradiation therapy had improved survival, suggesting some efficiency of this treatment on the corresponding target cells in these patients [36] (table 1). Larger series are needed to confirm these preliminary results, but there is evidence that this additional analysis of the conventional negative lymph nodes might lead to an improvement in tumor staging and help to select patients for adjuvant treatment. Detection of Disseminated Tumor Cells in Bone Marrow Disseminated tumor cells have been detected in the bone marrow of patients with various diseases although only a few of them are known to cause bone metastases. For patients with breast cancer a recently published study by Braun et al. [37] could demonstrate a significant prognostic difference between women with and without preoperative detection of disseminated tumor cells in their bone marrow by immunocytochemistry (antibody: A45B/B3). In pancreatic carcinoma patients disseminated tumor cells were detected by various immunocytological approaches (table 2). Detection rates of the preoperatively taken samples varied between 24 and 59% depending on the detection system and the tumor stages of the patients. Most of the studies found a significant difference of overall survival in univariate analysis between positive and negative patients, but so far none of the studies could demonstrate by multivariate analysis that disseminated tumor cells in bone marrow are an independent prognostic factor (table 2). Molecular biological studies of the bone marrow are rare. We found disseminated tumor cells in 19% of the bone marrow specimens of a small group of 27 patients with pancreatic carcinoma [38].
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In this now extended series of 88 bone marrow samples we detected in 11 patients (16%) with pancreatic carcinoma in UICC Stage I–IV a CK 20 signal [unpubl. data]. Detection of Disseminated Tumor Cells in Blood Since the development of PCR techniques venous blood of patients with pancreatic carcinoma has been analyzed in studies using markers such as K-ras, CEA, cytokeratin 19 and 20 (table 3). The detection rates of disseminated cells reached from 4 to 100% in peripheral venous blood depending on the used marker and the tumor stages of the patients. In none of the published studies, all of which included less than 50 patients each, univariate or multivariate analyses could be performed to answer the question of prognostic significance. Nomoto et al. [39] could demonstrate in a small group of 10 patients, that during operation tumor cells reach the peripheral blood in 50% using K-ras PCR. Aihara et al. [40] found in 2/49 patients a CK 19-positive signal also in the portal blood of patients with pancreatic carcinoma. A new approach by a combination of marker genes was published by Bilik et al. [41] who analyzed the mRNA expression of the tumor progression markers MET, GalNac-T and ß-hCG in various cell lines, tumor biopsies and the blood of 33 patients and healthy controls. They could demonstrate, that this approach leads to an increase in the overall detection rates. Only a very recently published study by Z’graggen et al. [42] tried to use immunostaining for the analysis of venous blood. They could demonstrate that the prevalence of disseminated tumor cells correlated with the UICC-tumor stage, but in the multivariate analysis no prognostic significance was observed (table 3). Detection of Disseminated Tumor Cells in Peritoneal Washings The detection of tumor cells in the peritoneal cavity of patients with pancreatic carcinoma is sometimes obvious due to the high rate of peritoneal carcinosis in these patients. But peritoneal carcinosis is often not detectable by the routinely performed tumor staging. Therefore preoperative laparoscopy is performed in some centers to detect small liver metastasis or peritoneal carcinosis. Peritoneal washings have sometimes been included and were first analyzed by conventional cytology [43]. Depending on the tumor stages disseminated tumor cells could be found in 7% up to 33% of the patients (table 4). Some of the studies demonstrated an influence on survival in univariate analysis, but only one multivariate analysis was performed [44], which could not demonstrate the detection of tumor cells as an independent factor. Further
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immunocytochemical analyses included mainly tumorassociated antibody combinations. Markary et al. [45] published a study on 137 pancreatic carcinoma patients and found an overall detection rate of 23% by cytology and immunocytology. A significant difference in survival was observed between patients with negative cytology and without visible metastases and patients with positive cytology without visible metastases in an univariate analysis. In a study of 80 patients, our group was able to demonstrate in a univariate analysis that the detection of disseminated tumor cells in the peritoneal cavity by using a cocktail of antibodies is of prognostic influence [46]. Only two molecularbiological analyses could demonstrate the detectability of cells in the peritoneal washing of patients with pancreatic carcinoma by PCR technique. Rall et al. [47] compared K-ras PCR and cytology. They detected cells in 2/24 patients by K-ras-specific PCR and in 3/24 patients with cytology, which demonstrated no benefit for the PCR-method (table 4). Approximately the same detection rate was observed by Inoue et al. [48]. They found 2 of 20 (10%) samples with K-ras expression. One aspect that has to be mentioned is the observation of higher detection rates in patients after percutaneous needle biopsy (28%) compared to only 17% in not-biopsied patients. Although this observation is not statistically significant, a trend towards tumor dissemination through biopsy techniques exists. These results give reason for caution against biopsy in potentially resectable patients with a possible increase of intraperitoneal tumor spread [49]. Detection of Disseminated Tumor Cells in Other Compartments Disseminated tumor cells can be found also in other compartments of patients with pancreatic carcinoma. Inoue et al. [48] demonstrated that in 13 of 17 (77%) patients with pancreatic carcinoma disseminated tumor cells could be detected in the liver by K-ras-specific PCR at the time of operation. Although only 3 of these patients had macroscopic metastases, in the other 10 patients with stage III tumors disseminated tumor cells had been observed. Suwa et al. [50] described a case of a woman with pancreatic cancer without visible liver metastases but positive K-ras signals in needle biopsies of her liver, who developed liver metastases during the follow-up. The same authors reported [51] nerve plexus invasions detected by immunostaining using an anti-cytokeratin 19 antibody and in parallel K-ras PCR in 17 cases of resected pan-
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creatic carcinoma. They found a neural invasion of the margin in 4 of 17 (23.5%) of the cases correlating significantly with a worth prognosis in univariate analysis. One additional patient was detected positive with the PCR methods. The analyses of pancreatic juice with the same methods mentioned so far has another aim. These analyses should lead to an earlier detection of pancreatic carcinoma especially to differentiate between patients with chronic pancreatitis and pancreatic carcinoma. Traditionally, cytological analyses have now been completed by p53 and K-ras mutation analyses. But these mutations can also be observed in chronic pancreatitis, although patients with K-ras mutations seem to have an increased risk to develop a pancreatic carcinoma [52, 53].
Conclusion and Perspectives
Despite the progress made in recent years, the prognosis for patients with pancreatic carcinoma remains poor, although more radical surgical approaches and multimodal therapeutic concepts are used. The main problem is the presence of early and occult tumor cell dissemination that leads to development of distant metastases. Based on the evaluation of the studies of breast cancer and other gastrointestinal carcinomas [33, 34, 37, 54], it can be assumed that disseminated tumor cells can also influence the survival of pancreatic carcinoma patients. However, an independent prognostic impact has to be sustained by further studies. One main problem that has to be solved is the missing standards for the detection methods followed by the difficulties to compare especially molecularbiological and immunohistochemical analyses. Molecular biological methods are much easier to standardize and less time-consuming compared to immunostaining but the high sensitivity of these techniques, ectopic nonfunctional expression of some possible target genes and the possible expression of pseudogenes may lead to false-positive results. So far, ideal marker genes that are tumor specific and expressed in every pancreatic carcinoma are lacking. Kras was thought to be a possible marker gene in pancreatic carcinoma because the frequency of the K-ras mutation is high (70–100%), but it was demonstrated that this mutation can also be observed in chronic pancreatitis and normal pancreatic tissue [20, 35]. CK 20 seems to be a better marker with a high expression in pancreatic carcinoma
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[55, 56], but as an epithelial marker it is alsoexpressed in other tumors and benign epithelial cells as well as in granulocytes [25]. Consequently, it is susceptible for contaminations of the investigated sample. New analyses of other mutations or combinations of genes that are specific for pancreatic carcinoma might lead to better candidates, and not only for the molecular biological methods. None of the antibodies used for immunostaining methods so far were found sufficient and demonstrated a high specificity. Combinations of antibodies are able to partially overcome the problem of heterogeneity and have reached the best results by immunostaining so far (tables 1–4). The advantage of immunocytochemistry is the chance to evaluate the morphology of the stained cells and therefore to detect false positive expression or cross-reactions and to count the number of positive cells, but the time-consuming evaluation needs an experienced interpreter even for computer-assisted evaluation methods. The development of quantitative PCR techniques also allows a calculation of the number of tumor cells in a sample. Because of their advantages, molecularbiological methods will become the methods of choice but their usefulness will be limited by the specificity of the marker genes. Beside the discussion on detection methods, we have to focus particularly on further methodical points like the sample size, cell separation methods and the compartments that are analyzed. Detection of disseminated tumor cells in lymph nodes of resected patients leads to an improvement of the local tumor staging, but hematogeneous tumor spread as detected in bone marrow or blood indicates a disseminated systemic disease. It is still a matter of controversy whether detection of tumor cells in the peritoneal cavity has the same prognostic relevance compared to findings in the
blood stream and whether disseminated tumor cells in bone marrow represent blood-borne tumor cells in transit or are trapped/filtered in this compartment with a likely consequence of a dormant state. These open questions have to be solved by comparative studies of paired samples from the same patients. The statistical analysis of the biological relevance of tumor cell detection in pancreatic carcinoma is additionally complicated by the generally short survival times implicating the need of high numbers of patients. Consequently, multicenter trials will be essential which, however, have to be performed with standardized surgical and detection procedures. Further studies and confirmations of the results achieved so far will lead to consequences for the indication of operations and resection techniques such as radical lymphadenectomy or preoperative standardized laparoscopy and peritoneal lavage [57]. The conventional staging nowadays clearly underestimates the tumor features regarding lymphatic but also hematogeneous spread. As recurrences are sometimes seen after a period of years, it is likely that disseminated cells can persist in a dormant state for prolonged periods of time. There is some evidence that these cells cannot be reached by conventional chemotherapy, but might be targeted by antibody-based adjuvant cancer therapies [58, 59]. One challenging problem for therapeutic strategies that has to be considered is the heterogeneity of solid tumors, since it drastically reduces the chance that all disseminated cells will be removed. An individualized characterization of the target cells may be attractive, but this does not appear applicable in a clinical setting. Therefore, cocktails of antibodies might be more efficient not only for diagnostic procedures but for therapeutic purposes as well.
References 1 Cameron JL, Pitt HA, Yeo CJ, Lillemore KD, Kaufmann HS, Coleman JA: One hundred and forty-five consecutive pancreaticoduodenectomies without mortality. Ann Surg 1993;217: 430–435. 2 Yeo CJ, Cameron LJ, Lillemoe KD, Sitzmann JV, Hruban RH, Goodmann SN, Dooley WC, Coleman JA, Pitt HA: Pancreaticoduodenectomy for cancer of the head on the pancreas: 201 patients. Ann Surg 1995;221:721–731.
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3 Bramhall SR, Allum WH, Jones AG, Allwood A, Cummins C, Neoptolemos JP: Treatment and survival in 13,560 patients with pancreatic cancer, and incidence of the disease, in the West Midlands: An epidemiological study. Br J Surg 1995;82:111–115. 4 Hosch SB, Knoefel WT, Metz S, Stoeklein N, Niendorf A, Broelsch CE, Itzbicki JR: Early lymphatic tumor cell dissemination in pancreatic cancer: Frequency and prognostic significance. Pancreas 1997;15:154–159.
5 Demeure MJ, Doffek KM, Komorowski RA, Wilson SD: Adenocarcinoma of the pancreas. Detection of occult metastases in regional lymph nodes by a polymerase chain reactionbased assay. Cancer 1998;83:1328–1334. 6 Brown HM, Ahrendt SA, Komorowski RA, Doffek KM, Wilson SD, Demeure MJ: Immunohistochemistry and molecular detection of nodal micrometastases in pancreatic cancer. J Surg Res 2001;95:141–146.
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7 Tamagawa E, Ueda M, Takahashi S, Sugano K, Uematsu S, Mukai M, Ogata Y, Kitajima M: Pancreatic lymph nodal and plexus micrometastases detected by enriched polymerase chain reaction and nonradioisotopic single strand conformation polymorphism analysis: A new predictive factor for recurrent pancreatic carcinoma. Clin Cancer Res 1997;3:2143–2149. 8 Ando N, Nakao A, Nomoto S, Takeda S, Kaneko T, Kurokawa T, Nonami T, Takagi H: Detection of mutant K-ras in dissected paraaortic lymph nodes of patients with pancreatic adenocarcinoma. Pancreas 1997;15:374–378. 9 Pantel K, Schlimok G, Angstwurm M, Weckermann D, Schmaus W, Gath H, Passlick B, Izbicki JR, Riethmüller G: Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 1994;3:165–173. 10 Traweek ST, Liu J, Battifora H: Keratine gene expression in non-epithelial tissues: Detection with polymerase chain reaction. Am J Pathol 1993;142:1111–1118. 11 Krüger W, Jung R, Kröger N, Gutensohn K, Fiedler W, Neumaier M, Jänicke F, Wagener C, Zander AR: Sensitivity of assays designed for the detection of disseminated epithelial tumor cells is influenced by cell separation methods. Clin Chem 2000;46:435–436. 12 Schlimok G, Funke I, Bock B, Schweiberer B, Witte J, Riethmüller G: Epithelial tumor cells in bone marrow of patients with colorectal cancer: Immunocytochemical detection, phenotypic characterization, and prognostic significance. J Clin Oncol 1990;8:831–837. 13 Juhl H, Stritzel M, Wroblewski A, HenneBruns D, Kremer B, Schmiegel W, Neumaier M, Wagener C, Schreiber HW, Kalthoff H: Immunocytochemical detection of micrometastatic ells: Comparative evaluation of findings in the peritoneal cavity and the bone marrow of gastric, colorectal and pancreatic cancer patients. Int J Cancer 1994;57:330–335. 14 Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, Duhig T, Peat N, Burchell J, Pemberton L, Lalini EN, Wuilson D: Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem 1996;265:15286–15293. 15 Schlimok G, Funke I, Holzmann B, Göttlinger G, Schmidt G, Häuser H, Swierkot S, Warnecke HH, Schneider B, Koprowski H, Riethmüller G: Micrometastatic cells in bone marrow: In vitro detection with anticytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies. Proc Natl Acad Sci USA 1987;84:8672–8676. 16 Brugger W, Bühring HJ, Grünebach F, Vogel W, Kraul S, Müller R, Brümmendorf TH, Ziegler BL, Rappold I, Brossart P, Scheding S, Kranz L: Expression of MUC-1 epitopes on normal bone marrow: Implications for the detection of micrometastatic tumor cells. J Clin Oncol 1999;17:1535–1544.
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17 Borgen E, Breiske K, Trachsel S, Nesland JM, Kvalheim G, Herstad TK, Schlichting E, Quist H, Naume B: Immunocytochemical detection of isolated epithelial cells in bone marrow: Non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase. J Pathol 1998;185:427–434. 18 Nicholson AG, Graham AN, Pezzella F, Agneta G, Goldstraw P, Pastorino U: Does the use of immunohistochemistry to identify micrometastases provide useful information in the staging of node-negative non-small cell lung carcinomas? Lung Cancer 1997;18:231–240. 19 Denis MG, Lipart C, Leborgne J, LeHur PA, Galmiche JP, Denis M, Ruud E, Truchaud A, Lustenberger P: Detection of disseminated tumor cells in peripheral blood of colorectal cancer patients. Int J Cancer 1997;74:540–544. 20 Löhr M, Maisonneuve P, Löwenfels AB: K-ras mutations and benign pancreatic disease. Int J Pancreatol 2000;27:93–103. 21 Soeth E, Röder C, Juhl H, Krüger U, Kremer B, Kalthoff H: The detection of disseminated tumor cells in bone marrow from colorectal-cancer patients by a cytokeratin-20-specific nested reverse-transcriptase-polymerase-chain reaction is related to the stage of disease. Int J Cancer 1996;69:278–282. 22 Schoenfeld A, Kruger KH, Gomm J, Sinnett HD, Gazet JC, Sacks N, Bender HG, Luqmani Y, Coombes RC: The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19. Eur J Cancer 1997;33:845–861. 23 Kwok S, Higuchi R: Avoiding false positives with PCR. Nature 1989;339:237–238. 24 Desol G, Gatter KC, Stein H, Erber WN, Pulford KAF, Zinnek B, Mason DY: Human lymphoid cells express epithelial membrane antigen: Implications for diagnosis of human neoplasms. Lancet 1984;11:1123–1129. 25 Jung R, Petersen K, Krüger W, Wolf M, Wagener C, Zander A, Neumaier M: Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes. Br J Cancer 1999;81:870–873. 26 Jung R, Krüger W, Hosch S, Holweg M, Kröger N, Gutensohn K, Wagener C, Neumaier M, Zander AR: Specificity of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro. Br J Cancer 1998;78:1194–1198. 27 Neumaier M, Gerhard M, Wagener C: Diagnosis of micrometastases by the amplification of tissue-specific genes. Gene 1995;159:43–47. 28 Goeminne JC, Guillaume T, Symann M: Pitfalls in the detection of disseminated nonhematological tumor cells. Ann Oncol 2000;11: 785–792. 29 Keilholz U, Willhauck M, Rimoldi D, Brasseur F, Dummer W, Rass K, de Vries T, Blaheta J, Voit C, Lethe B, Burchill S: Reliability of reverse transcription-polymerase chain reaction (RT-PCR)-based assays for the detection of circulating tumour cells: A quality-assurance
30
31
32
33
34
35
36
37
38
39
40
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initiative of the EORTC Melanoma Cooperative Group. Eur J Cancer 1998;34:750–753. Slade MJ, Smith BM, Sinnett HD, Cross NC, Coombes RC: Quantitative polymerase chain reaction for the detection of micrometastases in patients with breast cancer. J Clin Oncol 1999;17:870–879. Naume B, Borgen E, Nesland JM, Beiske K, Gilen E, Renolen A, Ravnas G, Quist H, Karesen R, Kvalheim G: Increased sensitivity for detection of micrometastases in bone marrow/ peripheral blood stem-cell products from breast-cancer patients by negative immunomagnetic separation. Int J Cancer 1998;78: 556–560. Gusterson BA, Gullick WJ, Venter DJ, Powles TJ, Elliott C, Ashley S, Tidy A, Harrison S: Immunohistochemical localization of c-erbB-2 in human breast carcinoma. Mol Cell Probes 1987;1:383–391. Raj GV, Moreno JG, Gomella LG: Utilization of polymerase chain reaction technology in the detection of solid tumors. Cancer 1998;82: 1419–1442. Burchill SA, Selby PJ: Molecular detection of low-level disease in patients with cancer. J Pathol 2000;190:6–14. Lüttges J, Schelche B, Menke MAOH, Vogel I, Henne-Bruns D, Klöppel G: The k-ras mutation pattern in pancreatic ductal adenocarcinoma is usually identical to that in associated normal, hyperplastic and metaplastic duct epithelium. Cancer 1999;85:1703–1710. Demeure MJ, Doffek KM, Komorowski RA, Redlich PN, Zhu Y, Erickson BA, Ritch PS, Pitt HA, Wilson SD: Molecular metastases in stage I pancreatic cancer: Improved survival with adjuvant chemoradiation. Surgery 1998; 124:663–669. Braun S, Pantel K, Müller P, Janni W, Hepp F, Kentenich CR, Gastroph S, Wischnik A, Dimpfl T, Kindermann G, Riethmüller G, Schlimok G: Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000;342:525–533. Soeth E, Vogel I, Röder C, Juhl H, Marxsen J, Krüger U, Henne-Bruns D, Kremer B, Kalthoff H: Comparative analysis of bone marrow and venous blood isolates from gastrointestinal cancer patients for the detection of disseminated tumor cells using reverse transcription PCR. Cancer Res 1997;57:3106–3110. Nomoto S, Nakao A, Kasai Y, Inoue S, Harada A, Nonami T, Takagi H: Peritoneal washing cytology combined with immunocytochemical staining and detecting mutant K-ras in pancreatic cancer: Comparison of the sensitivity and availability of various methods. Pancreas 1997;14:126–132. Aihara T, Noguchi S, Ishikawa O, Furukawa H, Hiratsuka M, Ohigashi H, Nakamori S, Monden M, Imaoka S: Detection of pancreatic and gastric cancer cells in peripheral and portal blood by amplification of keratin 19 mRNA with reverse transcriptase-polymerase chain reaction. Int J Cancer 1997;72:408–411.
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41 Bilchik A, Miyashiro M, Kelly M, Kuo C, Fujiwara Y, Nakamori S, Monden M, Hoon DS: Molecular detection of metastatic pancreatic carcinoma cells using a multimarker reverse transcriptase-polymerase chain reaction assay. Cancer 2000;88:1037–1044. 42 Z’graggen K, Centeno BA, Fernandez-del Castillo C, Jimenez RE, Werner J, Warshaw AL: Biological implications of tumor cells in blood and bone marrow of pancreatic cancer patients. Surgery 2001;129:537–546. 43 Warshaw AL: Implications of peritoneal cytology for staging of early pancreatic cancer. Am J Surg 1991;161:26–30. 44 Merchant NB, Conlon KC, Saigo P, Dougherty E, Brennan MF: Positive peritoneal cytology predicts unresectability of pancreatic adenocarcinoma. J Am Coll Surg 1999;188:421– 426. 45 Makary MA, Warshaw AL, Centeno BA, Willett CG, Rattner DW, Fernandez-del Castillo C: Implications of peritoneal cytology for pancreatic cancer management. Arch Surg 1998; 133:361–365. 46 Vogel I, Krüger U, Marxsen J, Soeth E, Kalthoff H, Henne-Bruns D, Kremer B, Juhl H: Disseminated tumor cells in pancreatic cancer patients detected by immunocytology: A new prognostic factor. Clin Cancer Res 1999;5: 593–599. 47 Rall CJ, Rivera JA, Centeno BA, Fernandezdel Castillo C, Rattner DW, Warshaw AL, Rustgi AK: Peritoneal exfoliative cytology and Ki-ras mutational analysis in patients with pancreatic adenocarcinoma. Cancer Lett 1995; 97:203–211. 48 Inoue S, Nakao A, Kasai Y, Harada A, Nonami T, Takagi H: Detection of hepatic micrometastases in pancreatic adenocarcinoma patients by two-stage polymerase chain reaction/restriction fragment length polymorphism analysis. Jpn J Cancer Res 1995;86:626–630. 49 Weiss SM, Skibber JM, Mohiuddin M, Rosato FE: Rapid intra-abdominal spread of pancreatic cancer. Influence of multiple operative biopsy procedures. Arch Surg 1985;120:415–416. 50 Suwa H, Hosotani R, Okino T, Monden K, Arii S, Inoue K, Kogire M, Ohshio G, Fukumoto M, Imamura M: Detection of multiple hepatic micrometastases in pancreatic adenocarcinoma with a solitary metastasis by direct sequencing of the k-ras gene: A case report. Surgery 1999; 125:113–116. 51 Suwa H, Hosotani R, Kogire M, Doi R, Ohshio G, Fukumoto M, Imamura M: Detection of extrahepatic nerve plexus invasion of pancreatic adenocarcinoma. Cytokeratin 19 staining and K-ras mutation. Int J Pancreatol 1999;26: 155–162. 52 Queneau PE, Adressi GL, Thibault P, Cleau H, Heyd B, Mantion G, Caraayon P: Early detection of pancreatic cancer in patients with chronic pancreatitis: Diagnostic utility of a Kras point mutation in the pancreatic juice. Am J Gastroenterol 2001;96:7000–7004.
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53 Müller P, Ostwald C, Puschel K, Brinkmann B, Platz F, Kroger J, Barten M, Nizze H, Schareck WD, Hauenstein K, Liebe S, Löhr JM: Low frequency of p53 and ras mutations in bile of patients with hepatobiliary disease: A prospective study in more than 100 patients. Eur J Clin Invest 2001;31:240–247. 54 Pantel K, Cote RJ, Fodstad Ø: Detection and clinical importance of micrometastatic disease. J Natl Cancer Inst 1999;91:1113–1124. 55 Wildi S, Kleeff J, Maruyama H, Maurer CA, Friess H, Büchler MW, Lander AD, Korc M: Characterization of cytokeratin 20 expression in pancreatic and colorectal cancer. Clin Cancer Res 1999;5:2840–2847. 56 Chu P, Wu E, Weiss LM: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: A survey of 435 cases. Mod Pathol 2000;13:962–972. 57 Jimenez RE, Warshaw AL, Fernandez-del Castillo C: Laparoscopy and peritoneal cytology in the staging of pancreatic cancer. J Hepatobiliary Pancreat Surg 2000;7:15–20. 58 Maxwell-Amstrong CA, Durrant LG, Scholefield JH: Immunotherapy for colorectal cancer. Am J Surg 1999;177:344–348. 59 Vermorken JB, Claessen AM, van Tinteren H, Gall HE, Ezinga R, Meijer S, Scheper RJ, Meijer CJ, Bloemena E, Ransom JH, Hanna MG Jr, Pinedo HM: Active specific immunotherapy for stage II and stage III human colon cancer: A randomised trial. Lancet 1999;353:345–350. 60 Thorban S, Roder JD, Pantel K, Siewert JR: Epithelial tumor cells in bone marrow of patients with pancreatic carcinoma detected by immunocytochemical staining. Eur J Cancer 1996;32A:363–365. 61 Thorban S, Roder JD, Pantel K, Siewert JR: Immunocytochemical detection of isolated epithelial tumor cells in bone marrow of patients with pancreatic carcinoma. Am J Surg 1996; 172:297–298. 62 Roder JD, Thorban S, Pantel K, Siewert JR: Micrometastases in bone marrow: Prognostic indicators for pancreatic cancer. World J Surg 1999;23:888–891. 63 Thorban S, Roder JD, Siewert JR: Detection of micrometastasis in bone marrow of pancreatic cancer patients. Ann Oncol 1999;10 (suppl 4):111–113. 64 Gerhard M, Juhl H, Kalthoff H, Schreiber HW, Wagener C, Neumaier M: Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol 1994;12:725–729. 65 Tada M, Omata M, Kawai S, Saisho H, Ohto M, Saiki RK, Sninsky JJ: Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma. Cancer Res 1993;53:2472–2474.
Pancreatology 2002;2:79–88
66 Funaki NO, Tanaka J, Kasamatsu T, Ohshio G, Hosotani R, Okino T, Imamura M: Identification of carcinoembryonic antigen mRNA in circulating peripheral blood of pancreatic carcinoma and gastric carcinoma patients. Life Sci 1996;59:2187–2199. 67 Nomoto S, Nakao A, Kasai Y, Harada A, Nonami T, Takagi H: Detection of ras gene mutations in perioperative peripheral blood with pancreatic adenocarcinoma. Jpn J Cancer Res 1996;87:793–797. 68 Chausovsky G, Luchansky M, Fier A, Shapira J, Gottfried M, Novis B, Bogelman G, Zemer R, Zimlichman S, Klein A: Expression of cytokeratin 20 in the blood of patients with disseminated carcinoma of the pancreas, colon, stomach and lung. Cancer 1999;86:2398–2405. 69 Kuroki T, Tomioka T, Tasjaima Y, Inoue K, Ikematsu Y, Ichinose K, Furui J, Kanematsu T: Detection of the pancreatic-specific gene in the peripheral blood of patients with pancreatic carcinoma. Br J Cancer 1999;81:350–353. 70 Miyazono F, Takao S, Natsugoe S, Uchikara K, Kijama F, Aridome K, Shinchi H, Aikou T: Molecular detection of circulating cancer cells in patients with biliary-pancreatic cancer. Am J Surg 1999;177:475–479. 71 Piva MG, Navaglia F, Basso D, Fogar P, Roveroni G, Gallo N, Zambon C-F, Pedrazzoli S, Plebani M: CEA mRNA identification in peripheral blood is feasible for colorectal, but not for gastric or pancreatic cancer staging. Oncology 2000;59:323–328. 72 Martin JK, Goellner JR: Abdominal fluid cytology in patients with gastrointestinal malignant lesions. Mayo Clin Proc 1986;61:467– 471. 73 Heeckt P, Safi F, Binder T, Bücheler M: Freie intraperitoneale Tumorzellen beim Pankreascarzinom – Bedeutung für den klinischen Verlauf und die Therapie. Chirurg 1992;63:563– 567. 74 Lei S, Kini J, Kim K, Howard JM: Pancreatic cancer: Cytologic study of peritoneal washings. Arch Surg 1994;129:639–642. 75 Leach SD, Rose JA, Lowy AM, Lee JE, Charnsangavej C, Abbruzzese JL, Katz RL, Evans DB: Significance of peritoneal cytology in patients with potentially resectable adenocarcinoma of the pancreatic head. Surgery 1995;118: 472–478. 76 Fernandez-del Castillo C, Rattner DW, Warshaw AL: Further experience with laparoscopy and peritoneal cytology in the staging of pancreatic cancer. Br J Surg 1995;82:1127–1129. 77 Nakao A, Oshima K, Takeda S, Kaneko T, Kanazumi N, Inoue S, Nomoto S, Kawase Y, Kusuya H: Peritoneal washing cytology combined with immunocytochemical staining in pancreatic cancer. Hepato-Gastroenterology 1999;46:2974–2977.
Vogel/Kalthoff/Henne-Bruns/Kremer
Detection and Prognostic Impact of Disseminated Tumor Cells in Pancreatic Carcinoma Ilka Vogel Holger Kalthoff Doris Henne-Bruns Bernd Kremer Molecular Oncology Research Laboratory, Department for General and Thoracic Surgery, University of Kiel, Germany
Key Words Disseminated tumor cells W Minimal residual disease W Pancreatic carcinoma
this increased staging has an prognostic impact and can be useful for therapeutic decisions in patients with pancreatic carcinoma. Copyright © 2002 S. Karger AG, Basel and IAP
Abstract Background/Aims: Metastatic disease determines the prognosis of patients with pancreatic cancer. Current routine staging methods often underestimate the tumor stage because they do not include the search for disseminated tumor cells that spread early in different compartments of the body. Immunohistochemical and molecular methods developed recently are able to detect these cells in multiple compartments of the body. Methods: The current status of the detection and the prognostic impact of disseminated tumor cells detected in lymph nodes, bone marrow, blood and peritoneal lavage of patients with pancreatic carcinoma are reviewed. Results: Disseminated tumor cells can be detected in different compartments of the body even in early tumor stages and when a resection of the primary tumor in curative intention was performed. Furthermore, the detection of these cells has importance for the prognosis and therefore will have therapeutic implications. Standardization of the methods is a prerequisite for further studies. Conclusion: The detection of disseminated tumor cells should be included into studies to reveal that
ABC
© 2002 S. Karger AG, Basel and IAP 1424–3903/02/0022–0079$18.50/0
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Accessible online at: www.karger.com/journals/pan
Introduction
The prognosis for patients with ductal pancreatic adenocarcinoma remains poor. Although operation techniques, multimodal treatment and lower postoperative mortality could improve survival, overall 5-year survival rates range between 5 and 20% [1, 2]. Only 10–20% of the patients diagnosed with pancreatic head carcinoma are resected with a curative intention [3]. But even in these patients tumor recurrence is frequent because of early lymphatic and haematogeneous distribution and consecutive development of distant metastases, local recurrence and peritoneal seeding. Assessment of lymph node status as an indicator for tumor dissemination is routinely done by histopathological examination. Tumor-associated antigens and/or tissue specific markers have been used to detect disseminated tumor cells in immunohistochemical analyses and have also improved the results of detection by conventional cytology [4]. Molecular biological approaches have been developed in recent years and focus mainly on
PD Dr. med. Ilka Vogel Klinik für Allgemeine Chirurgie und Thoraxchirurgie Forschungsgruppe Molekulare Onkologie, Universitätsklinikum Kiel Arnold-Heller-Strasse 7, D–24105 Kiel Tel. +49 431 597 4481, Fax +49 431 597 1939, E-Mail [email protected]
tumor-specific antigens and tissue-specific markers [5–8]. Additionally, bone marrow, blood and peritoneal washings have been analyzed (see tables 2–4). The studies demonstrate, that tumor staging can be improved by these methods but a definitive assessment of whether these cells are of prognostic relevance is complicated by the fact that many different methods and markers have been used with multiple detection systems. This review describes and summarizes the results of those studies, which analyzed disseminated tumor cells in lymph nodes, bone marrow, venous blood, peritoneal washings, and other compartments in patients with pancreatic carcinoma.
Results
Detection Methods – Methodical Aspects The detection of disseminated tumor cells depends on a number of steps including collection and treatment of the sample, cell separation protocol, chosen antibodies, number of analysed cells and evaluation techniques. All methods used depend on the recognition of antigens or gene transcripts that are believed to be exclusively expressed by tumor cells and not by surrounding cells in the examined compartment. In all methods (immunostaining or RT-PCR) unwanted positive results could occur due to contamination with skin cells, release of epithelial cells in benign proliferative diseases if epithelial markers are used [9, 10] or in case of an ectopic expression of epithelial markers in mesenchymal cells; false negative results may occur due to losses of tumor cells during isolation of mononuclear cells [11]. Immunostaining Approaches Immunostaining methods to detect epithelia-derived disseminated tumor cells in mesenchymal compartments like bone marrow exploit monoclonal antibodies directed against a variety of epithelia-specific cytoskeleton and membrane antigens (compare table 1–4). Several studies have clearly demonstrated that immunocytochemistry is superior to conventional cytology for the detection of isolated tumor cells in bone marrow [12] and peritoneal washings [13]. Pan-specific antibodies against cytokeratin seem to have a higher specificity than antibodies directed against the epithelial mucin family [14, 15]. Immunostaining lead to a further increase in sensitivity but a higher number of false positive results due to non-
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specific labeling were occasionally observed as well. The main reasons are: (i) expression of some epithelial antigens by a few hematological cells [16]; (ii) endogeneous alkaline phosphatase if anti-alkaline phosphatase (APAAP) complexes were used [17], and (iii) phagocytosed material in granulocytes than can be stained by antibodies [18]. Further investigations of new antibodies or combinations of antibodies that are highly specific and sensitive in their detection system are necessary. Depending on the detection system different authors have found in cell spiking experiments that it is possible to detect one tumor cell in 106 cells on a cytospin by immunocytochemical methods [9, 13]. This rate can be further increased by enrichment methods such as immunomagnetic beads isolation protocols [19]. To incorporate these methods into a clinical routine, it is necessary to standardize the methods used to date. Work on standardization of the evaluation of disseminated cells by immunohistochemical methods is currently in progress [17]. Molecular Biological Approaches Molecularbiological detection methods can be divided into two groups: (1) Detection of tumorspecific chromosomal abnormalities or mutations: In solid tumors the chromosomal abnormalities are heterogeneous and complex. An analysis of chromosomal abnormalities is therefore mainly restricted to tumors with mismatch-repair gene deficiencies. The use of specific mutations such as in the K-ras or p53-gene as markers is possible but the feasibility is rather poor [20]. (2) Detection of marker-gene expression (m-RNA) by reverse transcriptase polymerase chain reaction: Another way is the detection of RNA specific to the tissue of origin by reverse transcriptase polymerase chain reaction (RTPCR). Because mRNA is unstable in the extracellular environment, its detection in tissue or fluid is strongly indicative for the presence of vital tumor cells. For analyses of disseminated tumor cells with RNA-based assays for epithelial cells it has to be assumed that cells of nonhematopoietic origin are normally not circulating in the peripheral blood or bone marrow. The target RNA should not be expressed by hematopoietic cells, but by the disseminated tumor cells to be identified. Therefore, cytokeratins, especially cytokeratin 20 (CK 20) have been used (tables 2, 3) as epithelial-specific markers and the tumor-associated antigens like CEA and Ca 19-9 in pancreatic carcinoma. The PCR assays can detect a single target cell in up to 100 million irrelevant
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Table 1. Detection of disseminated tumor cells in lymph nodes of patients with pancreatic carcinoma Marker/antibodies
Detection rates
Patients R-situation
n
R0
18
Prognostic relevance univariate analysis
Author
multivariate analysis
Immunocytochemistry Ber-EP4
16/37 (43.2%) N0 lymphatic nodes of 13/18 patients (72.2%)
+ (overall and relapse-free survival)
Hosch et al. [4], 1997
Molecular biology K-ras
R0 10 UICC-stage I–III
4/6 patients (66.6%)
+
n.d.
Tamagawa et al. [7], 1997
K-ras (PCR/RFLP)
R0 (stage I/II)
15
8/13 (61.5%) 42/101 lymph nodes, paraaortic
n.d.
n.d.
Ando et al. [8], 1997
K-ras (PCR/RFLP)
R0 (stage I)
22
16/22 (73%) one or more regional lymph nodes
n.d.
n.d.
Demeure et al. [5], 1998
K-ras (PCR/RFLP)
R0 (stage I)
25
17/25 (68%) one or more regional lymph nodes positive
– n.d. 6/17 pos. patients and adju. therapy: +
Demeure et al. [36], 1998
AE3/AE1 (IHC)
R0 (stage I/II)
30
IHC: 14/30 (46.7%) PCR: 19/30 (63.3%) combination 25/30 (83.3%)
–
Brown et al. [6], 2001
K-ras (PCR/RFLP)
n.d.
–
+ = Relevant to prognosis; – = not relevant to prognosis; n.d. = not done.
Table 2. Detection of disseminated tumor cells in bone marrow samples of patients with pancreatic carcinoma Marker/antibodies
Detection rates
Patients R-situation
n
CEA, Ca 19-9, 17-1-A, C54-0, Ra 96, KL-1
R0-R2
32
CK2, KL-1, A45-B/B3
R0-R2
CK2, KL-1, A45-B/B3
CK2, KL-1, A45-B/B3
Prognostic relevance
Author
univariate analysis
multivariate analysis
15/26 (58%)
n.d.
n.d.
Juhl et al. [13], 1994
42
24/42 (57%)
+ (R0)
n.d.
Thorban et al. [60, 61], 1996
R0-R2
48
14/31 (48%) resected patients 10/18 (59%) not-resected patients
+
n.d.
Roder et al. [62], 1999
R0-R2
48
25/48 (52%) 4 (8.3%) CK2 positive 16 (33.3%) KL1 9 (18.6%) A45- B/B3 positive +
n.d.
Thorban et al. [63], 1999
Immunocytochemistry
CEA, Ca 19-9, 17-1-A, C54-0, Ra 96, KL-1
R0-R2
80
27/71 (38%)
(trend)
n.d.
Vogel et al. [46], 1999
AE1/AE3
R0-R2 54 (UICC-stage I–V)
13/54 (24%) R0: 13%
–
–
Z’graggen et al. [42], 2001
CEA
no details
3
2/3 (66%)
n.d.
n.d.
Gerhard et al. [64], 1994
CK 20
R0-R2
11
4/11 (36%)
n.d.
n.d.
Soeth et al. [21], 1996
CK 20
R0-R2
27 (BM)
5/27 (19%)
n.d.
n.d.
Soeth et al. [38], 1997
Molecular biology
+ = Relevant to prognosis; – = not relevant to prognosis; n.d. = not done.
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81
Table 3. Detection of disseminated tumor cells in blood samples of patients with pancreatic carcinoma Marker/antibodies
Detection rates
Patients R-situation
n
R0-R2 (stage I–IV)
105
R0–R2
6
Prognostic relevance
Author
univariate analysis
multivariate analysis
27/105 (26%) 3/32 (9%) R0 patients
–
–
Z’graggen et al. [42], 2001
2/6 (33.3%)
n.d.
n.d.
Tada et al. [65], 1993
Immunocytochemistry AE1/AE3
Molecular biology K-ras CEA
R0-R2
9
3/9 (33%)
n.d.
n.d.
Funaki et al. [66], 1996
K-ras
R0-R2
10
0/10 intraoperative 50% detection rate
n.d.
n.d.
Nomoto et al. [67], 1996
CK 19
no details
49
2/49 (4%) venous blood 2/49 (4%) portal blood
n.d.
n.d.
Aihara et al. [40], 1997
CK 20
R0-R2
22
2/22 (9%)
n.d.
n.d.
Soeth et al. [38], 1997
CK 20
R2 (stage IV)
28
22/28 (78%)
n.d.
n.d.
Chausovsky et al. [68], 1999
Chymotrypsinogen
R0-R2 (stage I–IV)
10
7/10 (70%)
n.d.
n.d.
Kuroki et al. [69], 1999
CEA
R0-R2 (stage I–IV)
21
13/21 (61.9%)
n.d.
n.d.
Miyazono et al. [70], 1999
MET, GalNac-T, ß-hCG
R0-R2
33
17/17 (100%) stage IV n.d. 8/16 (MET) 8/16 (GalNac-T) 7/16 (ß-hCG) 2/16 negative for all 3 markers in stage II/III
n.d.
Bilchik et al. [41], 2000
CEA
R0-R2
27
11/27 (41%)
n.d.
Piva et al. [71], 2000
n.d.
+ = Relevant to prognosis; – = not relevant to prognosis; n.d. = not done.
cells in spiking experiments [19, 21]. Direct comparison of a molecular biological with immunostaining methods showed that the PCR test is potentially 100 times more sensitive [22]. The high sensitivity and specificity of these assays can be limited, however, by DNA contamination [23], ‘illegitimate expression’ of epithelial or tumor-specific antigens in hematopoietic cells [16, 24, 25], upregulation of target mRNA expression by inflammation or hormonal induction of gene expression [26], the existence of pseudogenes that can lead to false-positive results in healthy individuals [25–27] or in patients with benign diseases [28], or the degradation of RNA during the various steps of the assay protocol [29]. Further studies should focus on quantitative RT-PCR which allows standardization of the amplification rate [30]. PCR-reactions with multiple markers may overcome tumor cell heterogeneity and false positive results and
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may yield an increased sensitivity and specificity. The enrichment of tumor cells, e.g. by magnetic beads, could solve the problem of false positives by reducing the background. Initial analyses with this method have been very promising [19, 31].
Results of the Clinical Studies and Prognostic Impact Detection of Disseminated Tumor Cells in Lymph Nodes It is generally accepted that lymph node metastases and therefore the UICC tumor stages have a prognostic value in most solid carcinoma. Conventional routine clinical histopathologic examination of regional lymph nodes is generally performed on formalin-fixed and paraffin-embedded tissue sections after staining with hematoxylin and eosin (HE). Mostly,
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Table 4. Detection of disseminated tumor cells in the peritoneal cavity of patients with pancreatic carcinoma Marker/antibodies
Detection rates
Patients R-situation
n
R0-R2
20
R0-R2
40
R0-R2
Prognostic relevance
Author
univariate analysis
multivariate analysis
5/25 (25%)
n.d.
n.d.
Martin et al. [72], 1986
12/40 (30%)
+
n.d.
Warshaw et al. [43], 1991
Cytology
57
12/36 (33%)
+
n.d.
Heeckt et al. [73], 1992
R0-R2 36 (stage I–IV)
3/36 (8.3%)
(+)
n.d.
Lei et al. [74], 1994
R0
60
4/60 (7%)
–
n.d.
Leach et al. [75], 1995
R0-R2
94
16/94 (17%)
n.d.
n.d.
Fernandez-del Castillo et al. [76], 1995
R0-R2 228 (stage I–IV)
34/228 (15%)
+
–
Merchant et al. [44], 1999
CEA, Ca 19-9, 17-1-A, C54-0, Ra 96, KL-1
R0-R2
31
18/31 (58%)
n.d.
n.d.
Juhl et al. [13], 1994
Ca 19-9, CEA
R0-R2
20
4/20 (20%)
n.d.
n.d.
Nomoto et al. [39], 1997
Ca 19-9, B 72. 3, CEA Leu-M1 and cytology
R0-R2
137
32/137 (23%)
(+)
n.d.
Makary et al. [45], 1998
CEA, Ca 19-9
R0-R2
74
8/8 (100%) peritoneal carcinosis 14/66 (22%) without visible peritoneal carcinosis
–
n.d.
Nakao et al. [77], 1999
CEA, Ca 19-9, 17-1-A, C54-0, Ra 96, KL-1
R0-R2
80
24/62 (39%)
+
n.d.
Vogel et al. [46], 1999
K-ras PCR
R0-R2
24
2/24 (8%) Cytology 3/24 (12%)
n.d.
n.d.
Rall et al. [47], 1995
K-ras PCR
R0-R2
20
2/20 (10%)
n.d.
n.d.
Inoue et al. [48], 1995 Nomoto et al. [39], 1997
Immunocytochemistry
Molecular biology
+ = Relevant to prognosis; (+) = relevant to prognosis of a subgroup; – = not relevant to prognosis; n.d. = not done.
only some representative sections of the many lymph nodes are examined which leads to underestimation of the stage. Gusterson et al. [32] calculated, for breast cancer lymph nodes, that histopathologic examination has only a 1% chance to identify a small lesion smaller than 3 cells. The analysis of more sections increase the detection rate, but also do not reach the detection rates observed by immunocytochemistry. Hosch et al. [4] found by the use of the pancytokeratin antibody Ber-EP4 in 16 of 37 conventional histologically negative lymph nodes disseminated tumor cells by immunostaining. They could also demonstrate an influence on overall and relapse-free survival in multivariate analysis when negative and positive tested patients were compared.
Molecular assays based on the PCR further increased the detection rates (tables 1–4) [33, 34], but because the tumor cells are not histologically identified or visualized, there are some concerns especially from traditional pathologists whether the detection of a gene product indicates the presence of a tumor cell. As up to 95% of the pancreatic carcinoma harbor a Kras point mutation [35], the presence of the mutant gene in a lymph node was interpreted as a proof for metastatic ells in the lymph node examined. The first study from Tamagawa et al. [7] demonstrated positive findings in 4 of 6 patients, who were classified as N0 by conventional analyses K-ras mutations. Ando et al. [8] and Demeure et al. [5, 36] confirmed these results with detection rates
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from 61.5% up to 73% in histological negative lymph nodes. In a recent study by Brown et al. [6], the nodes were analyzed by stepwise serial section and immunocytochemistry for cytokeratin (antibody: AE3/AE1) and K-ras PCR in parallel. By immunocytochemistry 46.7% of the conventional negative lymph nodes showed disseminated tumor cells and in 63.3% of the cases a K-ras analysis was positive. If both analyses were combined there was evidence for tumor cells in 83.3% of the conventionally negative nodes. However, this group found no difference in survival between the group with or without detected tumor cells. Patients with disseminated tumor cells who received adjuvant chemoradiation therapy had improved survival, suggesting some efficiency of this treatment on the corresponding target cells in these patients [36] (table 1). Larger series are needed to confirm these preliminary results, but there is evidence that this additional analysis of the conventional negative lymph nodes might lead to an improvement in tumor staging and help to select patients for adjuvant treatment. Detection of Disseminated Tumor Cells in Bone Marrow Disseminated tumor cells have been detected in the bone marrow of patients with various diseases although only a few of them are known to cause bone metastases. For patients with breast cancer a recently published study by Braun et al. [37] could demonstrate a significant prognostic difference between women with and without preoperative detection of disseminated tumor cells in their bone marrow by immunocytochemistry (antibody: A45B/B3). In pancreatic carcinoma patients disseminated tumor cells were detected by various immunocytological approaches (table 2). Detection rates of the preoperatively taken samples varied between 24 and 59% depending on the detection system and the tumor stages of the patients. Most of the studies found a significant difference of overall survival in univariate analysis between positive and negative patients, but so far none of the studies could demonstrate by multivariate analysis that disseminated tumor cells in bone marrow are an independent prognostic factor (table 2). Molecular biological studies of the bone marrow are rare. We found disseminated tumor cells in 19% of the bone marrow specimens of a small group of 27 patients with pancreatic carcinoma [38].
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In this now extended series of 88 bone marrow samples we detected in 11 patients (16%) with pancreatic carcinoma in UICC Stage I–IV a CK 20 signal [unpubl. data]. Detection of Disseminated Tumor Cells in Blood Since the development of PCR techniques venous blood of patients with pancreatic carcinoma has been analyzed in studies using markers such as K-ras, CEA, cytokeratin 19 and 20 (table 3). The detection rates of disseminated cells reached from 4 to 100% in peripheral venous blood depending on the used marker and the tumor stages of the patients. In none of the published studies, all of which included less than 50 patients each, univariate or multivariate analyses could be performed to answer the question of prognostic significance. Nomoto et al. [39] could demonstrate in a small group of 10 patients, that during operation tumor cells reach the peripheral blood in 50% using K-ras PCR. Aihara et al. [40] found in 2/49 patients a CK 19-positive signal also in the portal blood of patients with pancreatic carcinoma. A new approach by a combination of marker genes was published by Bilik et al. [41] who analyzed the mRNA expression of the tumor progression markers MET, GalNac-T and ß-hCG in various cell lines, tumor biopsies and the blood of 33 patients and healthy controls. They could demonstrate, that this approach leads to an increase in the overall detection rates. Only a very recently published study by Z’graggen et al. [42] tried to use immunostaining for the analysis of venous blood. They could demonstrate that the prevalence of disseminated tumor cells correlated with the UICC-tumor stage, but in the multivariate analysis no prognostic significance was observed (table 3). Detection of Disseminated Tumor Cells in Peritoneal Washings The detection of tumor cells in the peritoneal cavity of patients with pancreatic carcinoma is sometimes obvious due to the high rate of peritoneal carcinosis in these patients. But peritoneal carcinosis is often not detectable by the routinely performed tumor staging. Therefore preoperative laparoscopy is performed in some centers to detect small liver metastasis or peritoneal carcinosis. Peritoneal washings have sometimes been included and were first analyzed by conventional cytology [43]. Depending on the tumor stages disseminated tumor cells could be found in 7% up to 33% of the patients (table 4). Some of the studies demonstrated an influence on survival in univariate analysis, but only one multivariate analysis was performed [44], which could not demonstrate the detection of tumor cells as an independent factor. Further
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immunocytochemical analyses included mainly tumorassociated antibody combinations. Markary et al. [45] published a study on 137 pancreatic carcinoma patients and found an overall detection rate of 23% by cytology and immunocytology. A significant difference in survival was observed between patients with negative cytology and without visible metastases and patients with positive cytology without visible metastases in an univariate analysis. In a study of 80 patients, our group was able to demonstrate in a univariate analysis that the detection of disseminated tumor cells in the peritoneal cavity by using a cocktail of antibodies is of prognostic influence [46]. Only two molecularbiological analyses could demonstrate the detectability of cells in the peritoneal washing of patients with pancreatic carcinoma by PCR technique. Rall et al. [47] compared K-ras PCR and cytology. They detected cells in 2/24 patients by K-ras-specific PCR and in 3/24 patients with cytology, which demonstrated no benefit for the PCR-method (table 4). Approximately the same detection rate was observed by Inoue et al. [48]. They found 2 of 20 (10%) samples with K-ras expression. One aspect that has to be mentioned is the observation of higher detection rates in patients after percutaneous needle biopsy (28%) compared to only 17% in not-biopsied patients. Although this observation is not statistically significant, a trend towards tumor dissemination through biopsy techniques exists. These results give reason for caution against biopsy in potentially resectable patients with a possible increase of intraperitoneal tumor spread [49]. Detection of Disseminated Tumor Cells in Other Compartments Disseminated tumor cells can be found also in other compartments of patients with pancreatic carcinoma. Inoue et al. [48] demonstrated that in 13 of 17 (77%) patients with pancreatic carcinoma disseminated tumor cells could be detected in the liver by K-ras-specific PCR at the time of operation. Although only 3 of these patients had macroscopic metastases, in the other 10 patients with stage III tumors disseminated tumor cells had been observed. Suwa et al. [50] described a case of a woman with pancreatic cancer without visible liver metastases but positive K-ras signals in needle biopsies of her liver, who developed liver metastases during the follow-up. The same authors reported [51] nerve plexus invasions detected by immunostaining using an anti-cytokeratin 19 antibody and in parallel K-ras PCR in 17 cases of resected pan-
Disseminated Tumor Cells in Pancreatic Cancer
creatic carcinoma. They found a neural invasion of the margin in 4 of 17 (23.5%) of the cases correlating significantly with a worth prognosis in univariate analysis. One additional patient was detected positive with the PCR methods. The analyses of pancreatic juice with the same methods mentioned so far has another aim. These analyses should lead to an earlier detection of pancreatic carcinoma especially to differentiate between patients with chronic pancreatitis and pancreatic carcinoma. Traditionally, cytological analyses have now been completed by p53 and K-ras mutation analyses. But these mutations can also be observed in chronic pancreatitis, although patients with K-ras mutations seem to have an increased risk to develop a pancreatic carcinoma [52, 53].
Conclusion and Perspectives
Despite the progress made in recent years, the prognosis for patients with pancreatic carcinoma remains poor, although more radical surgical approaches and multimodal therapeutic concepts are used. The main problem is the presence of early and occult tumor cell dissemination that leads to development of distant metastases. Based on the evaluation of the studies of breast cancer and other gastrointestinal carcinomas [33, 34, 37, 54], it can be assumed that disseminated tumor cells can also influence the survival of pancreatic carcinoma patients. However, an independent prognostic impact has to be sustained by further studies. One main problem that has to be solved is the missing standards for the detection methods followed by the difficulties to compare especially molecularbiological and immunohistochemical analyses. Molecular biological methods are much easier to standardize and less time-consuming compared to immunostaining but the high sensitivity of these techniques, ectopic nonfunctional expression of some possible target genes and the possible expression of pseudogenes may lead to false-positive results. So far, ideal marker genes that are tumor specific and expressed in every pancreatic carcinoma are lacking. Kras was thought to be a possible marker gene in pancreatic carcinoma because the frequency of the K-ras mutation is high (70–100%), but it was demonstrated that this mutation can also be observed in chronic pancreatitis and normal pancreatic tissue [20, 35]. CK 20 seems to be a better marker with a high expression in pancreatic carcinoma
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85
[55, 56], but as an epithelial marker it is alsoexpressed in other tumors and benign epithelial cells as well as in granulocytes [25]. Consequently, it is susceptible for contaminations of the investigated sample. New analyses of other mutations or combinations of genes that are specific for pancreatic carcinoma might lead to better candidates, and not only for the molecular biological methods. None of the antibodies used for immunostaining methods so far were found sufficient and demonstrated a high specificity. Combinations of antibodies are able to partially overcome the problem of heterogeneity and have reached the best results by immunostaining so far (tables 1–4). The advantage of immunocytochemistry is the chance to evaluate the morphology of the stained cells and therefore to detect false positive expression or cross-reactions and to count the number of positive cells, but the time-consuming evaluation needs an experienced interpreter even for computer-assisted evaluation methods. The development of quantitative PCR techniques also allows a calculation of the number of tumor cells in a sample. Because of their advantages, molecularbiological methods will become the methods of choice but their usefulness will be limited by the specificity of the marker genes. Beside the discussion on detection methods, we have to focus particularly on further methodical points like the sample size, cell separation methods and the compartments that are analyzed. Detection of disseminated tumor cells in lymph nodes of resected patients leads to an improvement of the local tumor staging, but hematogeneous tumor spread as detected in bone marrow or blood indicates a disseminated systemic disease. It is still a matter of controversy whether detection of tumor cells in the peritoneal cavity has the same prognostic relevance compared to findings in the
blood stream and whether disseminated tumor cells in bone marrow represent blood-borne tumor cells in transit or are trapped/filtered in this compartment with a likely consequence of a dormant state. These open questions have to be solved by comparative studies of paired samples from the same patients. The statistical analysis of the biological relevance of tumor cell detection in pancreatic carcinoma is additionally complicated by the generally short survival times implicating the need of high numbers of patients. Consequently, multicenter trials will be essential which, however, have to be performed with standardized surgical and detection procedures. Further studies and confirmations of the results achieved so far will lead to consequences for the indication of operations and resection techniques such as radical lymphadenectomy or preoperative standardized laparoscopy and peritoneal lavage [57]. The conventional staging nowadays clearly underestimates the tumor features regarding lymphatic but also hematogeneous spread. As recurrences are sometimes seen after a period of years, it is likely that disseminated cells can persist in a dormant state for prolonged periods of time. There is some evidence that these cells cannot be reached by conventional chemotherapy, but might be targeted by antibody-based adjuvant cancer therapies [58, 59]. One challenging problem for therapeutic strategies that has to be considered is the heterogeneity of solid tumors, since it drastically reduces the chance that all disseminated cells will be removed. An individualized characterization of the target cells may be attractive, but this does not appear applicable in a clinical setting. Therefore, cocktails of antibodies might be more efficient not only for diagnostic procedures but for therapeutic purposes as well.
References 1 Cameron JL, Pitt HA, Yeo CJ, Lillemore KD, Kaufmann HS, Coleman JA: One hundred and forty-five consecutive pancreaticoduodenectomies without mortality. Ann Surg 1993;217: 430–435. 2 Yeo CJ, Cameron LJ, Lillemoe KD, Sitzmann JV, Hruban RH, Goodmann SN, Dooley WC, Coleman JA, Pitt HA: Pancreaticoduodenectomy for cancer of the head on the pancreas: 201 patients. Ann Surg 1995;221:721–731.
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3 Bramhall SR, Allum WH, Jones AG, Allwood A, Cummins C, Neoptolemos JP: Treatment and survival in 13,560 patients with pancreatic cancer, and incidence of the disease, in the West Midlands: An epidemiological study. Br J Surg 1995;82:111–115. 4 Hosch SB, Knoefel WT, Metz S, Stoeklein N, Niendorf A, Broelsch CE, Itzbicki JR: Early lymphatic tumor cell dissemination in pancreatic cancer: Frequency and prognostic significance. Pancreas 1997;15:154–159.
5 Demeure MJ, Doffek KM, Komorowski RA, Wilson SD: Adenocarcinoma of the pancreas. Detection of occult metastases in regional lymph nodes by a polymerase chain reactionbased assay. Cancer 1998;83:1328–1334. 6 Brown HM, Ahrendt SA, Komorowski RA, Doffek KM, Wilson SD, Demeure MJ: Immunohistochemistry and molecular detection of nodal micrometastases in pancreatic cancer. J Surg Res 2001;95:141–146.
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7 Tamagawa E, Ueda M, Takahashi S, Sugano K, Uematsu S, Mukai M, Ogata Y, Kitajima M: Pancreatic lymph nodal and plexus micrometastases detected by enriched polymerase chain reaction and nonradioisotopic single strand conformation polymorphism analysis: A new predictive factor for recurrent pancreatic carcinoma. Clin Cancer Res 1997;3:2143–2149. 8 Ando N, Nakao A, Nomoto S, Takeda S, Kaneko T, Kurokawa T, Nonami T, Takagi H: Detection of mutant K-ras in dissected paraaortic lymph nodes of patients with pancreatic adenocarcinoma. Pancreas 1997;15:374–378. 9 Pantel K, Schlimok G, Angstwurm M, Weckermann D, Schmaus W, Gath H, Passlick B, Izbicki JR, Riethmüller G: Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 1994;3:165–173. 10 Traweek ST, Liu J, Battifora H: Keratine gene expression in non-epithelial tissues: Detection with polymerase chain reaction. Am J Pathol 1993;142:1111–1118. 11 Krüger W, Jung R, Kröger N, Gutensohn K, Fiedler W, Neumaier M, Jänicke F, Wagener C, Zander AR: Sensitivity of assays designed for the detection of disseminated epithelial tumor cells is influenced by cell separation methods. Clin Chem 2000;46:435–436. 12 Schlimok G, Funke I, Bock B, Schweiberer B, Witte J, Riethmüller G: Epithelial tumor cells in bone marrow of patients with colorectal cancer: Immunocytochemical detection, phenotypic characterization, and prognostic significance. J Clin Oncol 1990;8:831–837. 13 Juhl H, Stritzel M, Wroblewski A, HenneBruns D, Kremer B, Schmiegel W, Neumaier M, Wagener C, Schreiber HW, Kalthoff H: Immunocytochemical detection of micrometastatic ells: Comparative evaluation of findings in the peritoneal cavity and the bone marrow of gastric, colorectal and pancreatic cancer patients. Int J Cancer 1994;57:330–335. 14 Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, Duhig T, Peat N, Burchell J, Pemberton L, Lalini EN, Wuilson D: Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem 1996;265:15286–15293. 15 Schlimok G, Funke I, Holzmann B, Göttlinger G, Schmidt G, Häuser H, Swierkot S, Warnecke HH, Schneider B, Koprowski H, Riethmüller G: Micrometastatic cells in bone marrow: In vitro detection with anticytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies. Proc Natl Acad Sci USA 1987;84:8672–8676. 16 Brugger W, Bühring HJ, Grünebach F, Vogel W, Kraul S, Müller R, Brümmendorf TH, Ziegler BL, Rappold I, Brossart P, Scheding S, Kranz L: Expression of MUC-1 epitopes on normal bone marrow: Implications for the detection of micrometastatic tumor cells. J Clin Oncol 1999;17:1535–1544.
Disseminated Tumor Cells in Pancreatic Cancer
17 Borgen E, Breiske K, Trachsel S, Nesland JM, Kvalheim G, Herstad TK, Schlichting E, Quist H, Naume B: Immunocytochemical detection of isolated epithelial cells in bone marrow: Non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase. J Pathol 1998;185:427–434. 18 Nicholson AG, Graham AN, Pezzella F, Agneta G, Goldstraw P, Pastorino U: Does the use of immunohistochemistry to identify micrometastases provide useful information in the staging of node-negative non-small cell lung carcinomas? Lung Cancer 1997;18:231–240. 19 Denis MG, Lipart C, Leborgne J, LeHur PA, Galmiche JP, Denis M, Ruud E, Truchaud A, Lustenberger P: Detection of disseminated tumor cells in peripheral blood of colorectal cancer patients. Int J Cancer 1997;74:540–544. 20 Löhr M, Maisonneuve P, Löwenfels AB: K-ras mutations and benign pancreatic disease. Int J Pancreatol 2000;27:93–103. 21 Soeth E, Röder C, Juhl H, Krüger U, Kremer B, Kalthoff H: The detection of disseminated tumor cells in bone marrow from colorectal-cancer patients by a cytokeratin-20-specific nested reverse-transcriptase-polymerase-chain reaction is related to the stage of disease. Int J Cancer 1996;69:278–282. 22 Schoenfeld A, Kruger KH, Gomm J, Sinnett HD, Gazet JC, Sacks N, Bender HG, Luqmani Y, Coombes RC: The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19. Eur J Cancer 1997;33:845–861. 23 Kwok S, Higuchi R: Avoiding false positives with PCR. Nature 1989;339:237–238. 24 Desol G, Gatter KC, Stein H, Erber WN, Pulford KAF, Zinnek B, Mason DY: Human lymphoid cells express epithelial membrane antigen: Implications for diagnosis of human neoplasms. Lancet 1984;11:1123–1129. 25 Jung R, Petersen K, Krüger W, Wolf M, Wagener C, Zander A, Neumaier M: Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes. Br J Cancer 1999;81:870–873. 26 Jung R, Krüger W, Hosch S, Holweg M, Kröger N, Gutensohn K, Wagener C, Neumaier M, Zander AR: Specificity of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro. Br J Cancer 1998;78:1194–1198. 27 Neumaier M, Gerhard M, Wagener C: Diagnosis of micrometastases by the amplification of tissue-specific genes. Gene 1995;159:43–47. 28 Goeminne JC, Guillaume T, Symann M: Pitfalls in the detection of disseminated nonhematological tumor cells. Ann Oncol 2000;11: 785–792. 29 Keilholz U, Willhauck M, Rimoldi D, Brasseur F, Dummer W, Rass K, de Vries T, Blaheta J, Voit C, Lethe B, Burchill S: Reliability of reverse transcription-polymerase chain reaction (RT-PCR)-based assays for the detection of circulating tumour cells: A quality-assurance
30
31
32
33
34
35
36
37
38
39
40
Pancreatology 2002;2:79–88
initiative of the EORTC Melanoma Cooperative Group. Eur J Cancer 1998;34:750–753. Slade MJ, Smith BM, Sinnett HD, Cross NC, Coombes RC: Quantitative polymerase chain reaction for the detection of micrometastases in patients with breast cancer. J Clin Oncol 1999;17:870–879. Naume B, Borgen E, Nesland JM, Beiske K, Gilen E, Renolen A, Ravnas G, Quist H, Karesen R, Kvalheim G: Increased sensitivity for detection of micrometastases in bone marrow/ peripheral blood stem-cell products from breast-cancer patients by negative immunomagnetic separation. Int J Cancer 1998;78: 556–560. Gusterson BA, Gullick WJ, Venter DJ, Powles TJ, Elliott C, Ashley S, Tidy A, Harrison S: Immunohistochemical localization of c-erbB-2 in human breast carcinoma. Mol Cell Probes 1987;1:383–391. Raj GV, Moreno JG, Gomella LG: Utilization of polymerase chain reaction technology in the detection of solid tumors. Cancer 1998;82: 1419–1442. Burchill SA, Selby PJ: Molecular detection of low-level disease in patients with cancer. J Pathol 2000;190:6–14. Lüttges J, Schelche B, Menke MAOH, Vogel I, Henne-Bruns D, Klöppel G: The k-ras mutation pattern in pancreatic ductal adenocarcinoma is usually identical to that in associated normal, hyperplastic and metaplastic duct epithelium. Cancer 1999;85:1703–1710. Demeure MJ, Doffek KM, Komorowski RA, Redlich PN, Zhu Y, Erickson BA, Ritch PS, Pitt HA, Wilson SD: Molecular metastases in stage I pancreatic cancer: Improved survival with adjuvant chemoradiation. Surgery 1998; 124:663–669. Braun S, Pantel K, Müller P, Janni W, Hepp F, Kentenich CR, Gastroph S, Wischnik A, Dimpfl T, Kindermann G, Riethmüller G, Schlimok G: Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000;342:525–533. Soeth E, Vogel I, Röder C, Juhl H, Marxsen J, Krüger U, Henne-Bruns D, Kremer B, Kalthoff H: Comparative analysis of bone marrow and venous blood isolates from gastrointestinal cancer patients for the detection of disseminated tumor cells using reverse transcription PCR. Cancer Res 1997;57:3106–3110. Nomoto S, Nakao A, Kasai Y, Inoue S, Harada A, Nonami T, Takagi H: Peritoneal washing cytology combined with immunocytochemical staining and detecting mutant K-ras in pancreatic cancer: Comparison of the sensitivity and availability of various methods. Pancreas 1997;14:126–132. Aihara T, Noguchi S, Ishikawa O, Furukawa H, Hiratsuka M, Ohigashi H, Nakamori S, Monden M, Imaoka S: Detection of pancreatic and gastric cancer cells in peripheral and portal blood by amplification of keratin 19 mRNA with reverse transcriptase-polymerase chain reaction. Int J Cancer 1997;72:408–411.
87
41 Bilchik A, Miyashiro M, Kelly M, Kuo C, Fujiwara Y, Nakamori S, Monden M, Hoon DS: Molecular detection of metastatic pancreatic carcinoma cells using a multimarker reverse transcriptase-polymerase chain reaction assay. Cancer 2000;88:1037–1044. 42 Z’graggen K, Centeno BA, Fernandez-del Castillo C, Jimenez RE, Werner J, Warshaw AL: Biological implications of tumor cells in blood and bone marrow of pancreatic cancer patients. Surgery 2001;129:537–546. 43 Warshaw AL: Implications of peritoneal cytology for staging of early pancreatic cancer. Am J Surg 1991;161:26–30. 44 Merchant NB, Conlon KC, Saigo P, Dougherty E, Brennan MF: Positive peritoneal cytology predicts unresectability of pancreatic adenocarcinoma. J Am Coll Surg 1999;188:421– 426. 45 Makary MA, Warshaw AL, Centeno BA, Willett CG, Rattner DW, Fernandez-del Castillo C: Implications of peritoneal cytology for pancreatic cancer management. Arch Surg 1998; 133:361–365. 46 Vogel I, Krüger U, Marxsen J, Soeth E, Kalthoff H, Henne-Bruns D, Kremer B, Juhl H: Disseminated tumor cells in pancreatic cancer patients detected by immunocytology: A new prognostic factor. Clin Cancer Res 1999;5: 593–599. 47 Rall CJ, Rivera JA, Centeno BA, Fernandezdel Castillo C, Rattner DW, Warshaw AL, Rustgi AK: Peritoneal exfoliative cytology and Ki-ras mutational analysis in patients with pancreatic adenocarcinoma. Cancer Lett 1995; 97:203–211. 48 Inoue S, Nakao A, Kasai Y, Harada A, Nonami T, Takagi H: Detection of hepatic micrometastases in pancreatic adenocarcinoma patients by two-stage polymerase chain reaction/restriction fragment length polymorphism analysis. Jpn J Cancer Res 1995;86:626–630. 49 Weiss SM, Skibber JM, Mohiuddin M, Rosato FE: Rapid intra-abdominal spread of pancreatic cancer. Influence of multiple operative biopsy procedures. Arch Surg 1985;120:415–416. 50 Suwa H, Hosotani R, Okino T, Monden K, Arii S, Inoue K, Kogire M, Ohshio G, Fukumoto M, Imamura M: Detection of multiple hepatic micrometastases in pancreatic adenocarcinoma with a solitary metastasis by direct sequencing of the k-ras gene: A case report. Surgery 1999; 125:113–116. 51 Suwa H, Hosotani R, Kogire M, Doi R, Ohshio G, Fukumoto M, Imamura M: Detection of extrahepatic nerve plexus invasion of pancreatic adenocarcinoma. Cytokeratin 19 staining and K-ras mutation. Int J Pancreatol 1999;26: 155–162. 52 Queneau PE, Adressi GL, Thibault P, Cleau H, Heyd B, Mantion G, Caraayon P: Early detection of pancreatic cancer in patients with chronic pancreatitis: Diagnostic utility of a Kras point mutation in the pancreatic juice. Am J Gastroenterol 2001;96:7000–7004.
88
53 Müller P, Ostwald C, Puschel K, Brinkmann B, Platz F, Kroger J, Barten M, Nizze H, Schareck WD, Hauenstein K, Liebe S, Löhr JM: Low frequency of p53 and ras mutations in bile of patients with hepatobiliary disease: A prospective study in more than 100 patients. Eur J Clin Invest 2001;31:240–247. 54 Pantel K, Cote RJ, Fodstad Ø: Detection and clinical importance of micrometastatic disease. J Natl Cancer Inst 1999;91:1113–1124. 55 Wildi S, Kleeff J, Maruyama H, Maurer CA, Friess H, Büchler MW, Lander AD, Korc M: Characterization of cytokeratin 20 expression in pancreatic and colorectal cancer. Clin Cancer Res 1999;5:2840–2847. 56 Chu P, Wu E, Weiss LM: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: A survey of 435 cases. Mod Pathol 2000;13:962–972. 57 Jimenez RE, Warshaw AL, Fernandez-del Castillo C: Laparoscopy and peritoneal cytology in the staging of pancreatic cancer. J Hepatobiliary Pancreat Surg 2000;7:15–20. 58 Maxwell-Amstrong CA, Durrant LG, Scholefield JH: Immunotherapy for colorectal cancer. Am J Surg 1999;177:344–348. 59 Vermorken JB, Claessen AM, van Tinteren H, Gall HE, Ezinga R, Meijer S, Scheper RJ, Meijer CJ, Bloemena E, Ransom JH, Hanna MG Jr, Pinedo HM: Active specific immunotherapy for stage II and stage III human colon cancer: A randomised trial. Lancet 1999;353:345–350. 60 Thorban S, Roder JD, Pantel K, Siewert JR: Epithelial tumor cells in bone marrow of patients with pancreatic carcinoma detected by immunocytochemical staining. Eur J Cancer 1996;32A:363–365. 61 Thorban S, Roder JD, Pantel K, Siewert JR: Immunocytochemical detection of isolated epithelial tumor cells in bone marrow of patients with pancreatic carcinoma. Am J Surg 1996; 172:297–298. 62 Roder JD, Thorban S, Pantel K, Siewert JR: Micrometastases in bone marrow: Prognostic indicators for pancreatic cancer. World J Surg 1999;23:888–891. 63 Thorban S, Roder JD, Siewert JR: Detection of micrometastasis in bone marrow of pancreatic cancer patients. Ann Oncol 1999;10 (suppl 4):111–113. 64 Gerhard M, Juhl H, Kalthoff H, Schreiber HW, Wagener C, Neumaier M: Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol 1994;12:725–729. 65 Tada M, Omata M, Kawai S, Saisho H, Ohto M, Saiki RK, Sninsky JJ: Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma. Cancer Res 1993;53:2472–2474.
Pancreatology 2002;2:79–88
66 Funaki NO, Tanaka J, Kasamatsu T, Ohshio G, Hosotani R, Okino T, Imamura M: Identification of carcinoembryonic antigen mRNA in circulating peripheral blood of pancreatic carcinoma and gastric carcinoma patients. Life Sci 1996;59:2187–2199. 67 Nomoto S, Nakao A, Kasai Y, Harada A, Nonami T, Takagi H: Detection of ras gene mutations in perioperative peripheral blood with pancreatic adenocarcinoma. Jpn J Cancer Res 1996;87:793–797. 68 Chausovsky G, Luchansky M, Fier A, Shapira J, Gottfried M, Novis B, Bogelman G, Zemer R, Zimlichman S, Klein A: Expression of cytokeratin 20 in the blood of patients with disseminated carcinoma of the pancreas, colon, stomach and lung. Cancer 1999;86:2398–2405. 69 Kuroki T, Tomioka T, Tasjaima Y, Inoue K, Ikematsu Y, Ichinose K, Furui J, Kanematsu T: Detection of the pancreatic-specific gene in the peripheral blood of patients with pancreatic carcinoma. Br J Cancer 1999;81:350–353. 70 Miyazono F, Takao S, Natsugoe S, Uchikara K, Kijama F, Aridome K, Shinchi H, Aikou T: Molecular detection of circulating cancer cells in patients with biliary-pancreatic cancer. Am J Surg 1999;177:475–479. 71 Piva MG, Navaglia F, Basso D, Fogar P, Roveroni G, Gallo N, Zambon C-F, Pedrazzoli S, Plebani M: CEA mRNA identification in peripheral blood is feasible for colorectal, but not for gastric or pancreatic cancer staging. Oncology 2000;59:323–328. 72 Martin JK, Goellner JR: Abdominal fluid cytology in patients with gastrointestinal malignant lesions. Mayo Clin Proc 1986;61:467– 471. 73 Heeckt P, Safi F, Binder T, Bücheler M: Freie intraperitoneale Tumorzellen beim Pankreascarzinom – Bedeutung für den klinischen Verlauf und die Therapie. Chirurg 1992;63:563– 567. 74 Lei S, Kini J, Kim K, Howard JM: Pancreatic cancer: Cytologic study of peritoneal washings. Arch Surg 1994;129:639–642. 75 Leach SD, Rose JA, Lowy AM, Lee JE, Charnsangavej C, Abbruzzese JL, Katz RL, Evans DB: Significance of peritoneal cytology in patients with potentially resectable adenocarcinoma of the pancreatic head. Surgery 1995;118: 472–478. 76 Fernandez-del Castillo C, Rattner DW, Warshaw AL: Further experience with laparoscopy and peritoneal cytology in the staging of pancreatic cancer. Br J Surg 1995;82:1127–1129. 77 Nakao A, Oshima K, Takeda S, Kaneko T, Kanazumi N, Inoue S, Nomoto S, Kawase Y, Kusuya H: Peritoneal washing cytology combined with immunocytochemical staining in pancreatic cancer. Hepato-Gastroenterology 1999;46:2974–2977.
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