Content uploaded by Peter J Hewett
Author content
All content in this area was uploaded by Peter J Hewett on Jul 01, 2020
Content may be subject to copyright.
MOLECULAR DETECTION OF BLOOD-BORNE EPITHELIAL CELLS
IN COLORECTAL CANCER PATIENTS AND IN PATIENTS
WITH BENIGN BOWEL DISEASE
Jennifer E. HARDINGHAM
1
*, Peter J. HEWETT
2
, Robert E. SAGE
1
, Jennie L. FINCH
1
, Jacqueline D. NUTTALL
1
, Dusan KOTASEK
1
and Alexander DOBROVIC
1
1
Department of Haematology-Oncology, The Queen Elizabeth Hospital, Woodville, South Australia, Australia
2
Department of Surgery, The Queen Elizabeth Hospital, Woodville, South Australia, Australia
In colorectal cancer (CRC), a proportion of patients with
early stage disease still die of metastatic or recurrent disease
within 5 years of “curative” resection. Detection of carci-
noma cells in the peripheral circulation at presentation may
identify a subgroup of patients with micro-metastatic disease
who may benefit from adjuvant chemotherapy or radiother-
apy. Our aim was to determine the presence and clinical
significance of colon carcinoma cells in peripheral blood at
the time of surgery. Preoperative peripheral blood samples
were collected from 94 patients with CRC and 64 patients
undergoing bowel resection for benign conditions (adenoma,
diverticular disease or Crohn’s colitis). Blood was also ob-
tained from 20 normal donors not undergoing bowel surgery.
Immunomagnetic beads were used to isolate epithelial cells
followed by reverse transcription-polymerase chain reaction
(RT-PCR) analysis of expression of cytokeratin (CK) 19, CK
20, mucin (MUC) 1 and MUC 2. Nineteen of 94 (20%) CRC
patients were positive for epithelial cells in preoperative
blood, including 6 with early stage disease. Kaplan–Meier
survival analysis showed that detection of epithelial cells in
preoperative blood was associated with reduced disease-free
and overall survival (log-rank test, pⴝ0.0001). Surprisingly,
circulating epithelial cells were detected in 3/30 (10%) pa-
tients resected for adenoma, and in 4/34 (12%) patients re-
sected for benign inflammatory conditions, suggesting that
cells from nonmalignant colonic epithelium may also gain
entry into the bloodstream in the presence of bowel pathol-
ogy. All 20 normal control bloods were negative for epithelial
cells. Int. J. Cancer (Pred. Oncol.) 89:8 –13, 2000.
©2000 Wiley-Liss, Inc.
Despite an apparently successful curative resection, about
10% of patients diagnosed with Dukes’ A and 30% with Dukes’
B colorectal tumours relapse and die of their disease within 5
years (American Joint Committee on Cancer, 1992). This find-
ing suggests that tumour cells with metastatic potential had
already escaped from the primary tumour before or at the time
of resection. Such cells may be present in the bloodstream in
very low numbers and would not be detected by conventional
means. The presence of tumour cells in the peripheral circula-
tion may be indicative of a micrometastatic process involving
distant organs and may have prognostic implications indepen-
dent of established staging factors such as the extent of lymph
node involvement.
A pilot study carried out in our unit of 27 colorectal cancer
(CRC) patients with K-ras mutant tumours, showed that immuno-
bead-PCR detection of K-ras mutant cells in peripheral blood (PB)
was significantly associated with reduced disease-free survival
(p⫽0.0001) (Hardingham et al., 1995). However, this technique
was applicable only in the 35% of our CRC patients whose tumour
carried the K-ras mutation.
To overcome the problem of tumour-specific mutations being
present in only a proportion of tumours, we sought to use reverse
transcription-polymerase chain reaction (RT-PCR) assays for the
expression of the epithelial-specific markers cytokeratin (CK) 19,
CK 20, mucin (MUC) 1 and MUC 2 to detect epithelial-derived
tumour cells in blood. CK 19 is a member of the intermediate
filament family of cytoskeletal proteins present in simple epithe-
lium such as breast and colon and their malignant counterparts
(Moll et al., 1982). CK 20 is another member of the intermediate
filament family and has been found to be a highly specific marker
for cells of the gastrointestinal tract (Moll et al., 1992). RT-PCR
for CK 19 expression was first developed by Datta et al. (1994) to
detect breast cancer cells in bone marrow, and adopted subse-
quently by others to detect epithelial-derived tumour cells in bone
marrow (BM), blood or lymph node tissue (reviewed by Raj et al.,
1998). RT-PCR for CK 20 expression has been used to detect
colon cancer cells in PB or BM (Burchill et al., 1995; Denis et al.,
1997; Gunn et al., 1996; Soeth et al., 1996). Importantly, neither
CK 19 nor CK 20 has been found to be expressed in skin epithe-
lium (Moll et al., 1982,
The study herein describes the use of immunobead RT-PCR
with a panel of epithelial-specific expression markers to iden-
tify epithelial cells in peripheral blood before surgery for pri-
mary CRC. Follow-up survival analysis shows a significant
correlation between the presence of marker-positive cells in the
circulation and reduced disease-free survival.We have also
shown, however, that colonic mucosal cells may be shed spon-
taneously into the circulation in cases of adenoma and inflam-
matory bowel disease.
MATERIAL AND METHODS
Patients
Ninety-four consecutive patients admitted for potentially cur-
ative resection of primary CRC were enrolled in the study.
Patients with overt metastatic disease were excluded. The me-
dian age of the patients was 72 years (range 32–99 years) with
a median follow-up of 445 days (range 16–1,870 days). Tu-
mours were staged according to the Dukes’ staging system.
Nonmalignant control subjects included 30 patients undergoing
resection for colorectal adenoma, 34 patients undergoing resec-
tion for inflammatory bowel disease (Crohn’s disease or diver-
ticulitis), and 20 healthy controls without bowel disease, not
undergoing surgery. Peripheral blood samples (20 ml) were
collected in dipotassium EDTA. Fresh tumour tissue was ob-
tained from CRC patients and snap frozen in liquid nitrogen for
RNA extraction. The study was approved by the Queen Eliza-
D. Kotasek’s present address is: Ashford Cancer Centre, Ashford, South
Australia 5035.
Grant sponsors: Anti-Cancer Foundation of the Universities of South
Australia; The Queen Elizabeth Hospital Research Foundation.
*Correspondence to: Haematology-Oncology Department, The Queen
Elizabeth Hospital, 28 Woodville Road, Woodville, South Australia 5011.
Fax: (61)8–82226144. E-mail: hardingham@tqehsmtp.tqeh.sa.gov.au
Received 25 March 1999; Revised 27 July 1999
Int. J. Cancer (Pred. Oncol.): 89, 8–13 (2000)
©2000 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
beth Hospital Ethics of Human Research Committee and in-
formed consent was obtained in all cases.
Cell lines
The colon cancer cell lines SW48, SW480, HT29, LIM-2412,
LIM-1215, LIM-2099, LIM-2405, LIM-1899, LIM-2463 and
LIM-1863 were tested by RT-PCR for expression of the 4 epithe-
lial-specific markers. Positive cell lines were used as controls and
to optimise the RT-PCR for each of the markers. The LIM cell
lines were kindly provided by Dr R. Whitehead (Ludwig Institute
for Cancer Research, Melbourne, Australia); the remainder were
purchased from the American Type Culture Collection (ATCC,
Rockville, MD). Cell lines were maintained in RPMI-1640 me-
dium with 10% fetal calf serum (FCS).
RNA extraction
RNA was extracted from tissue samples and cell lines using TRI
reagent (Sigma, St. Louis, MO) according to the manufacturer’s
protocol.
Design of primers
The primers for CK-19 were designed to avoid amplification of
the pseudogene (Datta et al., 1994) and do not amplify a product
from genomic DNA at the annealing temperature used (68°C) as
previously determined (Eaton et al., 1997). Primers for CK 20,
MUC 1 and MUC 2 were designed to span introns thus avoiding
amplification from genomic DNA. All primer pairs were checked
for their suitability using the program Amplify. Oligo probe se-
quences were selected to hybridise within the amplified product.
The sequences of primers and probes are shown in Table I.
Immunobead RT-PCR
The technique for immuno-magnetic isolation of epithelial cells
from blood has been described in detail previously (Hardingham,
1998). Briefly, each 10-mL blood sample was incubated with 3 ⫻
10
6
immuno-magnetic beads (Dynal, Oslo, Norway), labeled with
the epithelial-specific monoclonal antibody (MAb) Ber-EP4 (Da-
kopatts, Glostrup, Denmark) for 4 hr or overnight with gentle
mixing at room temperature. Bead-rosetted cells, isolated using a
magnetic array, were lysed in a volume of 15 L containing 0.1%
Nonidet P-40, 10 mM dithiothreitol (DTT) and 10 units RNasin
(Promega, Madison, WI) to release RNA (Eaton et al., 1997).
Following a 70°C denaturation for 3 min, reverse transcription was
carried out by the addition of 5⫻First Strand Buffer (250 mM
Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl
2
) 200 units of
M-MLV reverse transcriptase (both from Life Technologies, Be-
thesda, MD), 750 ng random hexamers (Pharmacia, Uppsala,
Sweden), 0.6 mM of each deoxynucleotide triphosphate (Roche,
Basel, Switzerland) and sterile ultra-pure water (RNAse-free)
(Biotech International, Perth, Australia) to a volume of 30 L. The
reaction was incubated at 37°C for 60 min.
Separate PCRs for the different markers were performed on a 7
L aliquot of cDNA in a final volume of 50 L, using the
following conditions: 100 ng of each primer, 0.75 units of Taq
polymerase (Amplitaq Gold, Perkin Elmer, Foster City, CA), 5 L
of 10⫻PCR buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15
mm MgCl
2
, 0.01% w/v gelatin) (Perkin Elmer), 200 M of each
deoxynucleotide triphosphate and sterile ultra-pure water. Cycling
parameters were as follows: an initial denaturation at 94°C for 5
min, then 1 min at 94°C, 68°C and 72°C for 45 cycles (CK 19 and
MUC 2), or 1 min at 94°C, 58°C, 72°C for 45 cycles (CK 20 and
MUC 1) with a final extension of 7 min at 72°C. Tubes spiked with
colon cancer cell line cells (100–200 cells) were used as positive
controls, whereas negative controls consisted of reagent only (no
target) RT and PCR tubes. Genomic DNA tubes were also in-
cluded to confirm that a signal was not produced from amplifica-
tion of genomic DNA. PCR products were run on a 1.5% agarose
gel, transferred to nylon membrane (Hybond N⫹, Amersham,
Aylesbury, UK), and hybridised to a
32
P end-labeled internal
oligo-probe. Autoradiographs were exposed for 6–72 hr.
Sensitivity experiments
The lower level of detection of tumour cells by immunobead
RT-PCR was determined by spiking 100, 10 and 0 colon cancer
cell-line cells into duplicate 10-mL samples of normal donor
blood.
Statistical analysis
Survival distributions were estimated using the product-limit
method of Kaplan and Meier (1958) and the log-rank test was used
to compare survival curves (Peto and Peto, 1972). Fisher’s exact
test was used to correlate the presence of positive cells in blood
with the extent of lymph node involvement in stage C patients. All
statistical calculations were performed using Graph Pad Prism
software.
RESULTS
Expression of epithelial-specific markers in primary tumours
and cell lines
To assess the suitability of the expression markers to detect
colon tumour cells, RT-PCR for each of the markers was per-
formed on RNA samples from primary colon tumours and colon
cancer cell lines. Thirteen of 14 tumours were positive for CK 19
(the negative sample was positive for the other 3 markers), 13/14
were positive for CK 20, 14/14 were positive for MUC 1 and 13/14
were positive for MUC 2 expression. In the colon cancer cell lines,
all 10 were positive for CK 19 expression, 9/10 were positive for
CK 20 (SW48 was negative), 8/10 were positive for MUC 1 (LIM
1863 and SW48 were negative) and 8/10 were positive for MUC
2 (SW48 and LIM 2405 were negative). The inability to amplify
FIGURE 1– Immunobead reverse transcription-polymerase chain re-
action (RT-PCR) sensitivity: autoradiograph showing results of im-
munobead RT-PCR for cytokeratin (CK) 20 after dilution of LIM-
2412 cells into duplicate 10-mL tubes of normal donor blood. Lane 1,
positive control (RT-PCR of 200 LIM-2412 cells in lysis mix); lanes
2 and 3, 0 cells in 10 mL blood; lanes 4 and 5, 10 cells in 10 mL blood;
lane 6, blank; lanes 7 and 8, 100 cells in 10 mL blood. The filter was
hybridised to a
32
P-labeled oligo-probe internal to the CK 20 primers.
TABLE I – SEQUENCES OF PRIMERS AND INTERNAL PROBES
Primer/probe Sequence 5⬘–3⬘
CK-19 sense GACTACAGCCACTACTACACGACC
CK-19 anti-sense AGCCGCGACTTGATGTCCATGAGCC
CK-19 internal probe GATCTGCATCTCCAGGTCGGTCC
CK-20 sense TCTTTGATGACCTAACCCTAC
CK-20 anti-sense CTACTTCTTGCACGACTGTCTTA
CK-20 internal probe AGCTCCGTTAGTTGAACCTCA
MUC 1 sense ACCAAGACTGATGCCAGTAGCACT
MUC 1 anti-sense ACCGTTACCTGCAGAAACCTTCT
MUC 1 internal probe CTATGAGAAGGTTTCTGCAGG
MUC 2 sense TGGCTGCGTGGTGGAGAAGGAA
MUC 2 anti-sense TTGGAGCAGGTGACGCCCGTAGT
MUC 2 internal probe TCAAGGTGGACTGCAATACCT
9CIRCULATING EPITHELIAL CELLS
CK 19, CK 20 or mucin transcripts in some tumours and cell lines,
despite successful amplification of other markers using the same
cDNA, indicated that a panel of markers was necessary. Appro-
priate cell lines served as positive controls in individual RT-PCR
assays.
Sensitivity experiments
In spiking experiments we were able to detect colon cancer
cell-line cells at the level of 10 cells per 10-mL blood sample (1
cell per mL, equivalent to 1 cell per 5–10 ⫻10
6
white cells). A
representative result is shown in Figure 1.
Immunobead RT-PCR analysis of blood samples
Nineteen of 94 (20%) CRC patients were positive for epithelial
RT-PCR markers in peripheral blood taken before surgical resec-
tion for malignancy. A representative result is shown in Figure 2.
Two of the 19 patients were staged as Dukes’ A, 4 as Dukes’ B and
13 as Dukes’ C (Table II). Among adenoma patients 3/30 were
positive, whereas 4/34 patients undergoing bowel resection for
benign conditions were positive (Table II). None of 20 normal
volunteers were positive for epithelial markers in blood.
Statistical analysis
Kaplan-Meier survival analysis of CRC patients was performed
using recurrence, development of metastases or death from disease
as endpoints. Data from 1 patient dying from other causes was
treated as a censored observation. The log-rank test was used to
compare survival curves of CRC patients positive or negative for
epithelial cells in blood. There was a significant difference be-
tween the 2 groups, those positive for epithelial markers in pre-
operative blood showing reduced time to relapse or death from
FIGURE 3– Kaplan-Meier survival analysis: comparison of survival
data from patients positive or negative for colon cells in blood: a, all
colorectal cancer patients, p⬍0.0001; b, Dukes’ stage C patients,
p⫽0.02.
FIGURE 2– Immunobead reverse transcription-polymerase chain reaction (RT-PCR) for cytokeratin (CK) 19: autoradiograph of controls and
patient blood samples. Lane 1, positive control cDNA (LIM-1215); lane 2, positive control cDNA (LIM-2412); lane 3, negative RT control (no
target); lane 4, negative PCR control (no target); lane 5, genomic DNA PCR control; lanes 6–13, patients’ preoperative blood samples. Note the
positive result in lane 6 (patient 473 in Table III).
TABLE II – PATIENTS POSITIVE FOR EPITHELIAL CELLS
IN PREOPERATIVE BLOOD
Dukes’ stage Number
of patients %
Positive
A(n⫽16) 2 12.5
B(n⫽47) 4 9
C(n⫽31) 13 42
Total n⫽94 19
Adenoma (n⫽30) 3 10
Benign (n⫽34) 4 12
Normal (n⫽18) 0
10 HARDINGHAM ET AL.
disease (p⬍0.0001, hazard ratio 3.9, 95% CI 2.99–21.34) (Fig.
3a).
There were 2 Dukes’ A patients who were positive for colon
cells in preoperative blood; 1 died of postoperative complications
at day 54, while the other was alive and disease free at last
follow-up (day 558). The 4 positive Dukes’ B patients remain
disease-free at median day 391 post-surgery (range 74–604). Of
13 Dukes’ C patients in whom circulating epithelial cells were
detected, 10 have died from metastatic disease, whereas another 3
are still alive following development of metastases. Kaplan–Meier
survival analysis for stage C patients showed a significant survival
advantage for patients who were negative for circulating epithelial
cells prior to surgery, compared with patients who were positive (p
⫽0.02, hazard ratio 2.8, 95% CI 1.169–6.716) (Fig. 3b). Recruit-
ment of further patients and longer follow-up is required to deter-
mine whether detection of blood-borne epithelial cells is indepen-
dent of tumour stage as a prognostic feature. However, within
stage C patients, detection of epithelial cells in PB was indepen-
dent of the extent of lymph node involvement (1–2 nodes vs. ⬎2
nodes, NS, Fisher’s exact test).
DISCUSSION
We have used the sensitive technique of immunobead RT-PCR
to detect epithelial cells in peripheral blood in patients about to
undergo surgical resection for colorectal malignancy or benign
bowel conditions. The prior immunobead isolation of epithelial
cells from blood enhances both the sensitivity and specificity of
RT-PCR detection and alleviates the need to use a two-stage
“nested” PCR to attain sensitivity which may result in detection of
very low level “illegitimate” transcription (Chelly et al., 1989).
The use of “nested” PCR has caused problems previously in
assessing the specificity of CK 19 as a marker for epithelial cells
(Krismann et al., 1995; Schoenfeld et al., 1994) but could be
overcome by using a one-stage PCR (Battaglia et al., 1998; Eaton
et al., 1997; Schoenfeld et al., 1994).
The fact that 20/20 normal control blood samples were negative
by immunobead RT-PCR for all 4 epithelial markers after South-
ern blot hybridisation and autoradiography, and that “spiked”
colon cancer cells were detectable at the level of 1 cell per 5–10 ⫻
10
6
white blood cells, indicates that this technique is both sensitive
and specific for epithelial cells. Whereas each of the markers
appears to be expressed in primary tumours and colon cancer cell
lines with about the same frequency, it is apparent that not all are
expressed at a similar level in any one tumour (Table III), empha-
sising the need to use a panel of markers to avoid false-negative
results.
In our study of CRC patients, the presence of epithelial-marker
positive cells in preoperative peripheral blood correlated with
reduced disease-free survival (p⬍0.0001), suggesting that at least
some of the cells detected were metastatic colon carcinoma cells in
transit in the circulation. Only a very small percentage of carci-
noma cells that intravasate into the bloodstream are able to form
metastases since most succumb to host immunological responses
or entrapment in the microvasculature (Weiss, 1990).
Among adenoma patients, 3/30 (all 3 diagnosed with large
tubular adenomas) were positive in preresection blood. One of
these patients (896, Table III) also had a needle biopsy of the
prostate performed on the day before blood collection, so that the
presence of CK 19 and CK 20 positive cells in the peripheral blood
might be explained by the biopsy procedure (transrectal) with or
without the release of prostatic epithelial cells.
Adenomatous tissue may contain foci of cells that have pro-
gressed toward a more malignant phenotype that may spontane-
ously shed cells into the circulation. However, 4/34 patients with
benign inflammatory conditions were also positive in preoperative
blood; the pathology in all 4 cases was described as diverticulitis;
in 1 of these patients there was severe inflammation and in another,
TABLE III – IMMUNOBEAD RT-PCR ANALYSIS: PATIENTS POSITIVE FOR EPITHELIAL CELLS IN PERIPHERAL BLOOD
Patient
ID Age
(years) Sex Dukes’
stage CK 19 CK 20 MUC 2 MUC 1
2
Follow-up
postresection
CRC
395 86 M A POS —
1
NEG — Died post-operative complications D 54
575 85 F A NEG NEG POS POS Alive disease free D 558
495 63 F B NEG NEG POS — NAD D 604
976 51 M B NEG NEG POS — NAD D 74
376 77 F B POS NEG NEG — Alive disease free D 527
581 79 F B NEG — POS — Alive disease free D 255
478 71 M C POS NEG NEG — Died recurrence D 395
334 56 M C NEG — POS — Died liver metastasis D 148
164 82 F C NEG — — POS Died liver metastasis D 793
488 83 M C POS — NEG — Alive with liver metastasis D 491
473 77 M C POS POS — NEG Died metastatic disease D355
486 79 M C POS — NEG — Died metastatic disease D 191
341 74 F C NEG POS NEG — Died metastatic disease D 542
117 57 F C NEG POS NEG — Alive, recurrent tumor D 785
530 66 M C POS POS NEG — Died metastatic disease D 345
625 82 M C POS — POS — Died disease D 16
195 78 M C POS — NEG — Died metastatic disease D 135
117 69 F C NEG POS NEG NEG Died metastatic disease D 130
218 60 F C NEG — POS — Died metastatic disease D 252
Adenoma
532 59 M NEG POS — — Right hemicolectomy, no recurrence D 425
896 81 M POS POS — — Right hemicolectomy, died met prostate ca D 385
988 76 F POS NEG POS — Transanal removal, no recurrence D 302
Benign
764 74 M NEG NEG POS — Subtotal colectomy, diverticulitis
914 55 F — NEG POS — Sigmoid colectomy, diverticulitis, micro-abscess
786 57 M NEG POS NEG NEG Sigmoid colectomy, diverticulitis
252 74 F NEG POS NEG NEG High anterior resection, diverticulitis
1
Not done; NAD, no abnormality detected.–
2
Fewer samples were tested using MUC 1 as this marker was introduced at a later stage of the
study.
11CIRCULATING EPITHELIAL CELLS
ulceration and perforation of the bowel wall. Perhaps inflammatory
changes and ulceration provide a means whereby normal colonic
mucosal cells gain access to the bloodstream.
Angiogenesis has been found to facilitate entry of tumour
cells into the circulation and has been found to be a predictor of
recurrence and survival in patients with early stage breast
(Weidner et al., 1992) and colon cancer (Frank et al., 1995).
Tumour-associated angiogenesis has also been found to occur
in adjacent normal mucosa (Fox et al., 1998) and may thus
provide a mechanism whereby normal colonic epithelial cells
gain access to the bloodstream. This mechanism may also be
relevant in the cases of large tubular adenoma in which angio-
genesis was present, as assessed by CD31 immunocytochemical
staining (data not shown).
Interestingly, the frequency of detection of circulating epithelial
cells in stage A or B patients was similar to that in patients with
adenoma or inflammatory bowel disease (Table II). None of the 5
early stage patients with circulating epithelial cells who were
followed have relapsed (Table III). Perhaps the cells detected were
normal colonic mucosal cells or tumour cells without metastatic
potential that had gained entry to the bloodstream via the newly
formed tumour-associated vasculature.
Few studies have correlated the finding of colon tumour cells in
peripheral blood with prognosis. Our earlier study of 27 patients
with K-ras mutation-positive tumours showed that patients in
whom circulating tumour cells were detected had a much poorer
prognosis than patients who were negative (p⫽0.0001) (Hard-
ingham et al., 1995). A study of 37 CRC patients, using CK 20 as
a marker for tumour cells in bone marrow and blood, found
significantly reduced survival in the bone marrow-positive group
(regardless of the blood status) compared with the group negative
in both compartments (Soeth et al., 1997). Another study used
RT-PCR to detect carcinoembryonic antigen (CEA) mRNA in
peripheral blood samples from patients with cancer, including 27
with CRC. Follow-up and survival analysis showed that the fre-
quency of cancer recurrence was significantly greater in CEA
mRNA-positive patients (Mori et al., 1998). Our present results
show, in a larger group of 94 CRC patients, that the presence of
epithelial marker-positive cells in peripheral blood before surgical
resection is predictive of shortened time to recurrence and overall
survival (p⬍0.0001).
Previously, epithelial-specific markers were considered ap-
propriate for analysing preoperative blood for the presence of
disseminated tumour cells as there was no published evidence
to suggest that normal colon cells have access to the blood-
stream in the absence of colorectal resection. We have now
shown that nonmalignant colonic mucosal cells may be shed
into the circulation in patients with adenoma or diverticulitis. It
is feasible, therefore, that normal colonic mucosal cells may
also be circulating in CRC patients and thus the finding of
blood-borne epithelial cells in individual CRC patients needs to
be interpreted with caution.
ACKNOWLEDGEMENTS
We thank Mrs. J.-X. Mi for excellent technical assistance, and
Ms. M. Colbeck and Mr. D. Beeslee for assistance with Cancer
Registry follow-up data.
REFERENCES
AMERICAN JOINT COMMITTEE ON CANCER,Manual for staging of cancer
(4th ed.), pp. 75–82, Lippincott, Philadelphia (1992).
BATTAGLIA, M., PEDRAZOZOLI, P., PALERMO, B., LANZA, A., BERTOLINI,
F., GIBELLI, N., DAPRADA, G.A., ZAMBELLI, A., PEROTTI,C.and
ROBUSTELLI DELLA CUNA, G., Epithelial tumor cell detection and the
unsolved problems of nested RT-PCR: a new sensitive one step method
without false positive results. Bone Marrow Transplant., 22, 693–698
(1998).
BURCHILL, S.A., BRADBURY, M.F., PITTMAN, K., SOUTHGATE, J., SMITH,B.
and SELBY, P., Detection of epithelial cancer cells in peripheral blood by
reverse transcriptase-polymerase chain reaction. Brit. J. Cancer, 71, 278–
281 (1995).
CHELLY, J., CONCORDET, J.P., KAPLAN, J.C. and KAHN, A., Illegitimate
transcription: transcription of any gene in any cell type. Proc. nat. Acad.
Sci. (Wash.), 86, 2617–2621 (1989).
DATTA, Y.H., ADAMS, P.T., DROBYSKI, W.R., ETHIER, S.P., TERRY, V.H.
and ROTH, M.S., Sensitive detection of occult breast cancer by the reverse-
transcriptase polymerase chain reaction. J. Clin. Oncol., 12, 475–482
(1994).
DENIS, M.G., LIPART, C., LEBORGNE, J., LEHUR, P.A., GALMICHE, J.P.,
DENIS, M., RUUD, E., TRUCHAUD, A. and LUSTENBERGER, P., Detection of
disseminated tumor cells in peripheral blood of colorectal cancer patients.
Int. J. Cancer, 74, 540–544 (1997).
DEVINE, P.L., BIRRELL, G.W., WHITEHEAD, R.H., HARADA, H., XING, P.X.
and MCKENZIE, I.F., Expression of MUC1 and MUC2 mucins by human
tumor cell lines. Tumor Biol., 13, 268–277 (1992).
EATON, M.C., HARDINGHAM, J.E., KOTASEK, D. and DOBROVIC, A., Immu-
nobead RT-PCR: a sensitive method for detection of circulating tumor
cells. Biotechniques, 22, 100–105 (1997).
FOX, S.H., WHALEN, G.F., SANDERS, M.M., BURLESON, J.A., JENNINGS, K.,
KURTZMAN, S. and KREUTZER, D., Angiogenesis in normal tissue adjacent
to colon cancer. J. Surg. Oncol., 69, 230–234 (1998).
FRANK, R.E., SACLARIDES, T.J., LEURGANS, S., SPEZIALE, N.J., DRAB, E.A.
and RUBIN, D.B., Tumor angiogenesis as a predictor of recurrence and
survival in patients with node-negative colon cancer. Ann. Surg., 222,
695–699 (1995).
GUNN, J., MCCALL, J.L., YUN, K. and WRIGHT, P.A., Detection of micro-
metastases in colorectal cancer patients by K19 and K20 reverse-transcrip-
tion polymerase chain reaction. Lab. Invest., 75, 611–616 (1996).
HARDINGHAM, J.E., Detection of circulating tumor cells using immuno-
bead-PCR. In: Y.M. Dennis Lo (ed.), Clinical applications of PCR, pp.
225–232, Humana Press, Totowa, NJ (1998).
HARDINGHAM, J.E., KOTASEK, D., SAGE, R.E., EATON, M.C., PASCOE, V.H.
and DOBROVIC, A., Detection of circulating tumor cells in colorectal cancer
by immunobead-PCR is a sensitive prognostic marker for relapse of
disease. Mol. Med., 1, 789–794 (1995).
HO, S.B., NIEHANS, G.A., LYFTOGT, C., YAN, P.S., CHERWITZ, D.L., GUM,
E.T., DAHIYA, R. and KIM, Y.S., Heterogeneity of mucin gene expression
in normal and neoplastic tissues. Cancer Res., 53, 641–651 (1993).
KAPLAN, E.L. and MEIER, P., Nonparametric estimation from incomplete
observations. J. Amer. Stat. Assoc., 53, 457–481 (1958).
KRISMANN, M., TODT, B., SCHRODER, J., GAREIS, D., MULLER, K.M.,
SEEBER, S. and SCHUTTE, J., Low specificity of cytokeratin 19 reverse
transcriptase-polymerase chain reaction analyses for detection of hematog-
enous lung cancer dissemination. J. Clin. Oncol., 13, 2769–2775 (1995).
MOLL, R., FRANKE, W.W., SCHILLE, R.D., GREIGER, B. and KREPLER, R.,
The catalog of human cytokeratins: patterns of expression in normal
epithelia, tumors and cultured cells. Cell, 31, 11–24 (1982).
MOLL, R., LOWE, A., LAUFER, J. and FRANKE, W.W., Cytokeratin 20 in
human carcinomas. A new histodiagnostic marker detected by monoclonal
antibodies. Amer. J. Pathol., 140, 427–447 (1992).
MORI, M., MIMORI, K., UEO, H., TSUJI, K., SHIRAISHI, T., BARNARD, G.F.,
SUGIMACHI, K. and AKIYOSHI, T., Clinical significance of molecular detec-
tion of carcinoma cells in lymph nodes and peripheral blood by reverse
transcription-polymerase chain reaction in patients with gastrointestinal or
breast carcinomas. J. Clin. Oncol., 16, 128–132 (1998).
NOGUCHI, S., AIHARA, T., NAKAMORI, S., MOTOMURA, K., INAJI, H.,
IMAOKA, S. and KOYAMA, H., The detection of breast carcinoma microme-
tastases in axillary lymph nodes by means of reverse transcriptase-poly-
merase chain reaction. Cancer, 74, 1595–1600 (1994).
PETO, R. and PETO, J., Asymptotically efficient rank invariant test proce-
dures (with discussion). J. Roy. Stat. Soc. A, 135, 185–189 (1972).
RAJ, G.V., MORENO, J.G. and LEONARD, G.G., Utilization of polymerase
chain reaction technology in the detection of solid tumours. Cancer, 82,
1419–1442 (1998).
SCHOENFELD, A., LUQMANI, Y., SMITH, D., O’REILLY, S., SHOUSHA, S.,
SINNETT, H.D. and COOMBES, R.C., Detection of breast cancer microme-
12 HARDINGHAM ET AL.
tastases in axillary lymph nodes by using polymerase chain reaction.
Cancer Res., 54, 2986–2990 (1994).
SOETH, E., RODER, C., JUHL, H., KRUGER, U., KREMER, B. and KALTHOFF,
H., The detection of disseminated tumor cells in bone marrow from
colorectal-cancer patients by a cytokeratin-20-specific nested reverse-tran-
scriptase-polymerase-chain reaction is related to the stage of disease. Int. J.
Cancer, 69, 278–282 (1996).
SOETH, E., VOGEL, I., RODER, C., JUHL, H., MARXSEN, J., KRUGER, U.,
HENNE-BRUNS, D., KREMER, B. and KALTHOFF, H., Comparative analysis of
bone marrow and venous blood isolates from gastrointestinal cancer pa-
tients for the detection of disseminated tumor cells using reverse transcrip-
tion PCR. Cancer Res., 57, 3106–3110 (1997).
WEIDNER, N., FOLKMAN, J., POZZA, F., BEVILACQUA, P., ALLRED, E.N.,
MOORE, D.H., MELI, S. and GASPARINI, G., Tumor angiogenesis: a new
significant and independent prognostic indicator in early-stage breast car-
cinoma [see comments]. J. nat. Cancer Inst. (Wash.), 84, 1875–1887
(1992).
WEISS, L., Metastatic inefficiency. Adv. Cancer Res., 54, 159–211 (1990).
13CIRCULATING EPITHELIAL CELLS
