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Chirurgische Klinik und Poliklinik [S. H., P. S., J. R. I., C. S.] and Molekulare Onkologie der Universitäts-Frauenklinik [K. P.], and Institut für Anatomie [U. S.], Universitätsklinikum Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany; Institut für Anthropologie und Humangenetik [J. K., M. R. S.] and Institut für Immunologie [K. W.], Ludwig-Maximilians-Universität München, Goethestrasse 31, D-80336 München, Germany
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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In the present investigation, we have analyzed 126 patients with completely resected esophageal cancer, and we provide evidence for a strong and independent prognostic influence of immunohistochemically identifiable tumor cells in lymph nodes. Because the direct analysis of these cells is hampered by their low frequency (10-4 to 10-5) we have used the latest culturing methods (5) to establish the first cell line derived from these cells. Subsequent analyses demonstrated, for the first time, that these cells are clonally selected viable tumor cells that are derived from the primary tumor and inherit a tumorigenic and metastatic potential in SCID4 mice.
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Patients and Methods |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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Lymph Node Preparation and Immunohistochemical Detection of
Tumor Cells
Lymph nodes, judged as tumor free by the surgeon, were divided
into two parts as described previously (6)
. One part of
each lymph node was embedded in paraffin for routine histopathological
staining; the other part were snap frozen in liquid nitrogen and stored
at -80°C for immunohistochemical analysis. For immunohistochemical
detection of tumor cells in lymph nodes, the anti-epithelial cell
monoclonal antibody Ber-EP4 (Dako, Hamburg, Germany) was used as
described previously (6
, 7)
.
Statistical Analysis
Associations between categorical parameters were assessed via
Fisher’s exact test. The Kaplan-Meier method was used to analyze local
recurrence-free, distant metastases-free, overall, and relapse-free
survival times. For comparison purposes, log-rank tests were performed.
Cox’s proportional hazards models were fitted for multivariate
analysis (8)
. Relative risk and 95% confidence limits are
presented. Differences between groups are considered significant if the
Ps were <0.05 for a two-tailed test.
Analysis of Genomic Changes, Tumorigenic and Micrometastatic
Potential of Immunohistochemically Identifiable Cells
Case Summary.
Cells from a primary tumor classified as poorly differentiated
adenocarcinoma and from a corresponding histopathologically tumor-free
lymph node were sampled for cell culturing for a detailed analysis. The
patient’s history can be summarized as follows. The patient underwent
radical en bloc esophagectomy with curative intention. In
routine histopathological examination, the surgical specimen had
tumor-free resection margins (R0), and the tumor was staged as
pT1pN1M0. In 2 of 38 resected lymph
nodes, metastases were found on routine H&E staining.
Immunocytochemical analysis of bone marrow aspirates obtained at the
time of primary surgery with monoclonal antibody A45B/B3 revealed no
evidence for hematogeneous tumor cell dissemination into this organ,
which has been implicated as indicator site for blood-borne metastatic
cells in esophageal cancer (9)
. One and a half months
after surgery, the patient was readmitted to the hospital because of a
large tumor mass in the mediastinum with malignant pleural effusion and
a portsite metastasis in the cutis, where a drainage had been placed
during primary surgery. No further treatment was endeavored, and the
patient died 2 months after primary surgery.
Generation of Cell Lines from mAb Ber-EP4-positive Cells and the
Primary Tumor.
Part of one lymph node, derived from the arteria gastrica sinistra and
judged as tumor free by H&E staining, and samples of the primary tumor
were harvested for culturing. These samples were cut into small
fragments, vigorously washed with RPMI 1640 (Life Technologies,
Paisley, Scotland) and disaggregated into single-cell suspensions using
the Medimachine (Dako, Hamburg, Germany). Cell suspensions were plated
in ECM-coated T25 culture flasks (Paesel and Lorei, Frankfurt, Germany)
and cultured as described previously (5)
.
DNA Analyses of Cell Lines.
Microsatellite analysis and HLA-DRB1* genotyping confirmed that both
cell lines were derived from the same patient. Genomic DNA was isolated
from cell lines following the protocol of Miller et al.
(10)
. The aqueous DNA solution was frozen at -20°C and
thawed for subsequent microsatellite analysis using the GenePrint
Fluorescent CTTv STR Multiplex System (Promega Corp., Madison, WI). In
addition, genomic DNA was used for HLA-DRB1* genotyping
(11)
.
For p53 sequence analysis of the primary tumor and lymph node
micrometastatic cell lines, total RNA was isolated by the method of
Chirgwin (12)
and purified to poly-A-RNA using the
Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany). The reverse
transcription of poly-A-RNA was performed using the Superscript II
reverse transcriptase (Life Technologies, Inc., Eggenstein, Germany)
and random hexameric primers according to the manufacturer’s
instructions. The primers 5'-TTTCCA CGA CGG TGA CAG C-3' (nucleotides
154-173) and 5'-CTG TCA TCC AAG TAC TCC ACA CGC G-3' (nucleotides
840-818) as well as 5'-ATG AGC GC TGC TCA GAT AGC-3' (nucleotides 720)
and 5'-AAG ACC CAA AACCCA AAA TGG-3' (nucleotides 1475-1455) were used
to amplify p53 cDNA. Depending on the primers used, the entire 1316-bp
p53 coding region, or two overlapping fragments which spanned the open
reading frame of the p53 cDNA, were obtained. Direct sequencing of PCR
products was performed with 100 ng (
0.25 pmol) of purified DNA and
10 pmol of the respective primer. DNA sequencing was performed by the
nonradioactive cycle sequencing method using the Taq-DNA polymerase
sequencing kit and dye-labeled dideoxynucleotides on a 377 stretch
system (Applied Biosystems, Inc., Weiterstadt, Germany) together with a
thermocycler (Model 9600; Perkin Elmer, Überlingen, Germany).
FISH.
M-FISH (13)
was done as described previously
(14)
. In brief, flow-sorted whole chromosome painting
probes (kindly provided by Prof. M. Ferguson-Smith, Cambridge, United
Kingdom) were amplified by DOP-PCR (15)
. Probe
labeling was performed in a stringent DOP-PCR assay. The probe mix was
hybridized for 2 days, and detection, image capturing, and image
processing were performed as described previously (14)
.
Results are displayed either as true color, generated simply by
overlaying the five source images (Fig. 3A)
, or
classification color (Fig. 3
, B, D, and E, third
column). FISH with CEPH-YAC 933a5 (kindly provided by Dr. T. Haaf,
MPI Berlin, Germany), mapped to 8q24.2-3 (16)
, was
hybridized as described before (17)
.
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Results |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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. There was no correlation
between the presence of Ber-EP4-positive cells in lymph nodes and the
histopathological stage (pT), type, or differentiation grade (G) of the
primary tumor (Table 1)
. As expected, Ber-EP4-positive cells were more
frequently detected in patients with overt lymph node metastatases (pN1
group) compared with those patients without such metastases (pN0 group;
Table 1
). In the group of 54 patients classified as pN0,
immunohistochemical analyses revealed monoclonal antibody
Ber-EP4-positive isolated cells in 30 (56%) patients. For survival
analysis, six patients were excluded because they died during their
hospital stays (within 60 days after surgery) because of postoperative
complications (e.g., respiratory failure, sepsis). From the
remaining 48 patients, 20 Ber-EP4-negative-pN0 patients had
a median relapse free survival of >55 months compared with 27 months
in the 28 Ber-EP4-positive-pN0 patients
(P = 0.005; Fig. 1
). Most remarkably, only 1 of 20
patients who were found to be free of nodal tumor involvement, as
assessed by both histopathological and immunohistochemical analysis,
developed recurrence during the observation period compared with 10 of
28 patients with a positive immunohistochemical finding. Similarly,
only 1 of 20 patients with Ber-EP4-negative nodes developed recurrence
and died, whereas 10 of 28 patients with Ber-EP4-positive nodes
relapsed, and 9 of these patients died during the observation period.
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Multivariate Cox regression analysis revealed an independent prognostic
influence of immunohistochemically detectable tumor cells in lymph
nodes for both relapse-free survival (P = 0.01) and overall survival (P = 0.02).
Furthermore, histopathological lymph node status was also of
independent prognostic significance for relapse-free and overall
survival, whereas primary tumor stage and grade had no independent
influence on patients prognosis (Table 2)
.
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. A cell line, designated
as LN1590, was generated from one of these Ber-EP4-positive nodes that
contained 3 Ber-EP4-positive cells per 105 lymph node
cells. Another cell line, designated as PT1590, was generated from the
autologous primary tumor using identical culture conditions. Both cell
lines have grown by now >50 passages and can therefore be classified
as immortal.
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A at position 738, resulting in a histidine to
arginine exchange (His176Arg176; data not
shown).
Both tumor cell lines were in the near-triploid range. Examples
of representative M-FISH karyotypes are shown in Fig. 3
A. Both cell lines shared
some common structural rearrangements including del(5)
(q1?3q2?2),
der(8)
, der(9)
del(9)
(:p2122q2122:), der(17)
,
der(22)
t(19;22), and loss of the Y chromosome (Fig. 3, A and B)
. An 8q24.2-3 band-specific YAC yielded signals that were
detected on both ends of the der(8)
(Fig. 3C)
. Together with
the 4',6-diamidino-2-phenylindole bands, the structure of the
der(8)
was determined as der(8)
(qterq1?::p2?qter). Further
aberrations were observed exclusively either in the primary tumor cells
or in the micrometastatic cells. In the primary tumor cells, five
structural changes, der(1)
t(1;20), der(3)
t(3;17), der(8)
t(5;8),
der(10)
t(1;10), and der(13)
t(5;13), could be observed in 50% of
metaphase spreads, which were not seen in the micrometastatic cells
(Fig. 3D)
. Only the micrometastatic cells showed an
insertion of chromosome 13 material in the short arm of chromosome 1,
resulting in a der(1)
ins(1;13)(p22;q?) (Fig. 3E)
. In
addition, some structural aberrations were observed in both cell lines
that occurred in a single metaphase only (data not shown).
Tumorigenic and Micrometastatic Potential of Immunohistochemically
Identifiable Cancer Cells in SCID Mice.
To assess the in vivo tumorigenic potential of LN1590 cells,
between 1 x 105 and 6.5 x 106 cells were transplanted s.c. into the lateral flanks of
immunodeficient SCID mice. After 2–29 weeks of observation (mean, 13.8
weeks), 12 of 13 mice developed local tumors at the site of injection
(Fig. 2B)
. In two animals, the local tumors invaded through
the peritoneum into the abdominal cavity. Macroscopic metastases into
the lung were detected in 5 animals. Furthermore, we were able to
re-establish tumor cell lines from different murine tissues, such as
mesenterial lymph nodes, bone marrow, and lung, despite the lack of
visible signs of metastases. This observation indicates the presence of
occult micrometastases in these organs.
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Discussion |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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The published data about cytogenetic changes in esophageal cancer are
limited, but recent comparative genomic hybridization studies indicated
8q as the most common region of gain and 5q12-21 as the most common
region of loss (18)
. The 8q overrepresentation correlates
well with the der(8)
(qterq1?::p2?qter), and the 5q loss
correlates with the del(5)
(q1?3q2?2) observed in both cell lines,
PT1590 and LN1590. Recently, a gain of 8q23–qter was even associated
with lymph node metastasis (19)
. Other findings, such as
the involvement of the short arm of chromosome 22 as seen in the
der(22)
t(19;22) or the loss of the Y chromosome, also fit very well
with observations published previously by others (18
, 20
, 21)
.
It is obvious that this approach has the potential to unravel loci in the genome, which are specifically associated with metastasis. The insertion of 13q material into the short arm of chromosome 1 seen only in the lymph node micrometastases but not in the primary tumor is intriguing because 1p was reported as frequently involved in structural rearrangements in esophageal cancers (20) . This suggests that cells carrying this aberration might have been selected during the process of lymphatic dissemination. The presence of such a micrometastasis-specific anomaly supports the model of clonal metastasis, which implicates that one tumor cell clone disseminates because of a decisive event discriminating it from the other primary tumor cells. After its arrival at the secondary site (e.g., lymph node), this clone needs to undergo further genomic instability to evolve into an overt metastasis.
Our results support the concept that our cell lines are a reliable in vitro system to analyze chromosome-specific aberrations of esophageal cancer and minimal residual disease in particular.
In vitro evidence that occult tumor cells are viable cells with an unlimited proliferative potential is already provided by the generation of such a cell line by itself. To demonstrate whether these cells are tumorigenic in vivo, the cultured cells were transplanted s.c. into immunodeficient SCID mice. Progressive tumor nodules were observed in most of the transplanted animals, proving that the cultured cells were indeed malignant tumor cells.
This is the first report demonstrating that occult tumor cells, detectable with immunohistochemical assays but undetectable by histopathological analyses, are tumorigenic and metastatic in vivo, which implies that these cells might be precursors of subsequent metastatic lesions rather than shedded tumor cells with a limited life span. The controversial discussions about the clinical and biological relevance of micrometastatic tumor cells in lymph nodes of patients with solid epithelial tumors may merely be attributable to assay variability. The Ber-EP4 antibody used in our present study was selected because it is homogeneously expressed on the majority of primary esophageal cancers, and it does not cross-react with normal lymph node cells (7) . In contrast, anti-cytokeratin antibodies, frequently used to detect such micrometastases (1) , bind to normal lymph node cells (reticulum cells) and therefore cause false-positive findings. This limited specificity might explain why Glickman et al. (22) failed to demonstrate an independent prognostic impact of cytokeratin-positive cells in lymph nodes of esophageal cancer patients. On the other hand, Luketich et al. (23) used CEA mRNA as marker for esophageal tumor cells and found a significant association to an unfavorable prognosis. Thus, there is an urgent need for standardization of the current protocols before micrometastatic lymph node staging can be implemented into clinical practice.
In summary, we demonstrated that immunohistochemically identifiable tumor cells present in lymph nodes judged as "tumor free" in routine histopathology are of independent strong predictive value for tumor relapse and overall survival in patients with esophageal cancer. Our observation should have important consequences for tumor staging and therapy. The presence of Ber-EP4-positive cells in lymph nodes should be incorporated in the UICC staging nomenclature by including micrometastases (addition: mi) and isolated tumor cells (addition: i) in the N-category of the Tumor-Node-Metastasis classification. Patients with occult dissemination of viable tumor cells are not cured by surgery alone and may benefit from additional adjuvant therapy. Interestingly, the antibody Ber-EP4, used for tumor cell detection in our study, recognizes the 17-1A antigen (also called epithelial cell adhesion molecule; Ref. 24 ), which is a promising target for antibody therapy in patients with solid epithelial tumors (1 , 25 , 26) .
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FOOTNOTES |
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1 This study was supported by grants of the
Deutsche Krebshilfe Mildred Scheel Stiftung, the Alfried Krupp von
Bohlen und Halbach-Stiftung, Hamburger Krebsgesellschaft and
Otto-Stiftung Hamburg, and a grant cofunded by Micromet GmbH, Munich,
and the Bundesministerium für Bildung und Forschung, Germany. 
2 These authors contributed equally.
3 To whom requests for reprints should be
addressed, at Frauenklinik, Universitatsklinikum, Eppendorf,
Martinistrasse 52, D-20246 Hamburg, Germany. Phone: 49 40 42803 3503;
Fax: 49 40 42803 5379; E-mail: pantel{at}uke.uni-hamburg.de
4 The abbreviations used are: SCID, severe
combined immunodeficient; FISH, fluorescence in situ hybridization;
M-FISH, multiplex fluorescence in situ hybridization.
Received 8/25/00. Accepted 10/17/00.
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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