Molecular Detection of Neoplastic Cells in Lymph Nodes of

Metastatic Colorectal Cancer Patients Predicts Recurrence

Montserrat Sanchez-Cespedes, Manel Esteller, Kenji Hibi, et al.

Clin Cancer Res 1999;5:2450-2454.

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2450 Vol. 5, 2450 –2454, September 1999
Molecular Detection of Neoplastic Cells in Lymph Nodes of
Metastatic Colorectal Cancer Patients Predicts Recurrence1
Montserrat Sanchez-Cespedes, Manel Esteller,
Kenji Hibi, Frederick O. Cope,
William H. Westra, Steven Piantadosi,
James G. Herman, Jin Jen, and David Sidransky2
Department of Otolaryngology-Head and Neck Surgery, Division of
Head and Neck Cancer Research [M. S-C., K. H., J. J., D. S.], Tumor
Biology [M. E., J. G. H.), Pathology Department [W. H. W.], and
Oncology Biostatistics [S. P.], The Johns Hopkins Oncology Center,
Baltimore, Maryland 21205, and Neoprobe Corp., Dublin, Ohio
[F. O. C.]
ABSTRACT
Disseminated disease, especially to the liver, constitutes
the major risk of recurrence for colorectal cancer patients.
However, successful resection can still be achieved in 25–
35% of colorectal cancer patients with isolated metastases.
To evaluate the clinical value of occult micrometastatic dis-
ease detection in lymph nodes, we tested genetic (K-ras and
p53 gene mutations) and epigenetic (p16 promoter hyper-
methylation) molecular markers in the perihepatic lymph
nodes from colorectal cancer patients with isolated liver
metastases. DNA was extracted from 21 paraffin-embedded
liver metastases and 80 lymph nodes from 21 colorectal
cancer patients. K-ras and p53 gene mutations were identi-
fied in DNA from liver metastases by PCR amplification
followed by cycle sequencing. A sensitive oligonucleotide-
mediated mismatch ligation assay was used to search for the
presence of K-ras and p53 mutations to detect occult disease
in 68 lymph nodes from tumors positive for these gene
mutations. Promoter hypermethylation at the p16 tumor
suppressor gene was examined in both liver lesions and
lymph nodes by methylation-specific PCR. Sixteen of the 21
(76%) liver metastases harbored either gene point mutations
or p16 promoter hypermethylation. Twelve of the 68 lymph
nodes were positive for tumor cells by molecular evaluation
and negative for tumor cells by histopathology and cyto-
keratin immunohistochemistry, whereas none were positive
for tumor cells by histopathology or negative for tumor cells
Received 3/5/99; revised 5/4/99; accepted 5/12/99.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
Supported in part by GI Specialized Program of Oncology Research
Excellence (SPORE) Grant CA 62924. M. S-C. and M. E. are recipients
of Spanish Ministerio de Educacion y Cultura Awards. J. J. and J. G. H.
are recipients of the V-Foundation Awards for Cancer Research.
2
To whom requests for reprints should be addressed, at Department of
Otolaryngology-Head and Neck Surgery, 818 Ross Research Building,
720 Rutland Avenue. Baltimore, MD 21205-2196. Phone: (410) 502-
5153; Fax: (410) 614-1411; E-mail: dsidrans@jhmi.edu.
Downloaded from clincancerres.aacrjournals.org on August 19, 2014. © 1999 American Association for Cancer
Research.
Clinical Cancer Research
by molecular analysis (P 5 0.0005, McNemar’s test). More-
over, in three patients with lymph nodes that were histolog-
ically negative at all sites, molecular screening detected tu-
mor DNA at one or more lymph nodes. Survival analysis
showed a median survival of 1056 days for patients without
evidence of lymph node involvement by molecular analysis
and 165 days for patients with positive lymph nodes by this
approach (P 5 0.0005). These results indicate that lymph
node metastasis screening in colorectal cancer patients by
molecular-based techniques increases the sensitivity of tu-
mor cell detection and can be a good predictor of recurrence
in colorectal cancer patients with resectable liver metastases.
INTRODUCTION
Colorectal cancer constitutes a major public health problem.
This disease affects about 1 in 20 people in Western countries, and
more than 155,000 new cases are diagnosed in the United States
each year. Overall, 25% of the patients with resectable disease at
the time of the diagnosis will have recurrent disease, presumably
from local, regional, and peritoneal seeding. Major improvements
in surgical procedures have allowed tumor resection in patients
with isolated liver, lung, or pelvic metastases. However, only
25–30% of patients achieve a 5-year disease-free survival.
There is not much information on the prognosis factors of this
subset of colorectal cancer patients. Perihepatic lymph node
involvement has been associated with a poor prognosis in
patients with isolated liver metastases (1), indicating the need
for improved control of micrometastases.
Genetic-based techniques have been used to detect micro-
metastases in regional lymph nodes and were reported to be
more sensitive than standard histopathology (2). Although sev-
eral genetic techniques have been used, they are all based on the
ability to perform PCR on tissue samples. Hayashi et al. (3)
have detected a small number of cancer cells by screening for
K-ras gene alterations with the mutant allele-specific amplifi-
cation analysis in lymph nodes negative for tumor cells by
morphological analysis. Other studies have used the reverse
transcription-PCR technique to detect gene expression for the
presence or absence of a specific tumor marker (4 – 6). More-
over, it has been reported that the detection of micrometastases
by molecular-based techniques in morphologically negative
lymph nodes is a prognostic tool in stage II colorectal cancer
patients (5, 7), suggesting that it could serve as a selective
marker for intensive postoperative adjuvant chemotherapy.
We have used a mismatch ligation assay to screen for
K-ras and p53 gene mutations (8) and the MSP3 technique (9)
to look for p16 promoter hypermethylation in the perihepatic
lymph nodes from colorectal cancer patients who underwent
3
The abbreviation used is: MSP, methylation-specific PCR.

liver metastasis resection. The purpose of our work was to
compare the sensitivity of our approach with standard histopa-
thology and to evaluate the clinical value of identifying positive
perihepatic lymph nodes by both morphological and molecular
methods to predict recurrence in colorectal cancer patients with
liver metastases.
MATERIALS AND METHODS
Patients. Twenty-one liver metastases and 80 lymph
nodes from 21 colorectal cancer patients were collected from
different hospitals in a multicenter study conducted by Neo-
probe Corp. designed to analyze the clinical relevance of the use
of radioimmunoguided surgery in the detection of lymph nodes
in metastatic colorectal cancer patients. All of the patients gave
written consent, and the study was approved by each local
institutional review board.
DNA Extraction. Paraffin blocks from the liver metas-
tases and the corresponding perihepatic lymph nodes were ob-
tained from each patient. The paraffin blocks were cut into
several 6-mm sections. A section from a liver metastasis was
stained with H&E for histopathological diagnosis to differenti-
ate neoplastic cells from their normal counterparts. DNA was
then extracted from unstained sections of the malignant tissue.
For cytokeratin immunohistochemical staining, 5-mm sections
from the paraffin blocks were stained with the monoclonal
antibody AE1:AE3 (Boehringer Mannheim, Indianapolis, IN;
1:2000 dilution) using the avidin-biotin-peroxidase technique.
Tissue sections were deparaffinized using 100% xylene fol-
lowed by 100% ethanol. The pellet was then resuspended in a
buffer containing SDS-proteinase K, and DNA was extracted
with phenol-chloroform followed by ethanol precipitation. Fi-
nally, precipitated DNA was resuspended in 100 ml of Tris-
EDTA buffer as described previously (10). DNA was quantified
spectrophotometrically, and 100 ng were used as a template for
each PCR amplification.
Mutation Detection. Exon 1 of K-ras and four individ-
ual exons of p53 (exons 5– 8) were amplified by PCR from liver
metastasis DNA. All PCR products were purified and sequenced
directly using the AmpliCycle sequencing kit (Perkin-Elmer,
Foster City, CA). The primers for the PCR and the sequencing
conditions used for each exon are described elsewhere (8). The
lymph nodes from those tumors positive for any mutation were
tested using a mismatch ligation assay. The mismatch ligation
procedure has been described previously (8). Briefly, the exon
containing the mutation was amplified by PCR, and the product
was purified before the mismatch ligation assay. Each PCR
product (50 ng) was mixed with 8 ng of a common 32P-labeled
oligomer in a 20-ml reaction containing 150 mM NaCl, 10 mM
MgCl2, 100 mM Tris-HCl (pH 7.5), 1 mM spermidine, 1 mM
DTT, 1 mM ATP, and 3 mg of T4 gene 32 protein (Boehringer
Mannheim). This mixture was denatured at 95°C for 5 min and
allowed to cool at room temperature for 15 min, at which time
1 unit of T4 ligase was added. The ligation was carried out at
37°C for 1 h and terminated by heat inactivation at 68°C for 10
min. The [32P]phosphate on the unligated 39 oligomer was
removed by the addition of 1 unit of alkaline phosphatase and
subsequent incubation at 37°C for 30 min. The ligation products
were then separated on a 12% denaturing polyacrylamide gel.
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Clinical Cancer Research 2451
Table 1 Molecular alterations detected in liver metastases from
colorectal cancer patients
Molecular alteration No. of tumors (%) Detection method
K-ras mutations 10/21 (48%) Cycle sequencing
p53 mutations 6/21 (29%) Cycle sequencing
p16 methylation 3/21 (14%) MSP
Total 16/21 (76%) Any
The corresponding mutation in the tumor DNA was used as a
positive control, and a negative tumor for the mutation was used
as a negative control in each reaction. Oligonucleotide se-
quences for the K-ras ligation assay have been described pre-
viously (11). Specific oligomers were designed individually to
identify p53 gene mutations (8).
MSP. For p16 promoter hypermethylation analysis, 1 mg
of DNA was denatured by NaOH and then modified by sodium
bisulfite. DNA samples were then purified using Wizard DNA
purification resin (Promega, Madison, WI), treated again with
NaOH, precipitated with ethanol, and resuspended in water.
PCR was performed separately with methylation-specific prim-
ers and nonmethylation primers for each tumor sample (9).
Controls without DNA and positive controls for unmethylated
and methylated reactions were performed for each set of PCR
reactions. PCR products were analyzed on nondenaturing 6%
polyacrylamide gels, stained with ethidium bromide, and visu-
alized under UV illumination.
Statistical Analysis. The cumulative survival rates for
patients groups were calculated using the Kaplan-Meier method
(12) and compared using the log-rank test. To evaluate the
difference in sensitivity between histopathological and molecu-
lar lymph node metastasis detection, we used McNemar’s test.
All reported Ps are two-sided.
RESULTS
Identification of Genetic Alterations at the Liver Me-
tastasis Tissue. We identified either K-ras and p53 gene
mutations or p16 gene promoter hypermethylation at 16 of the
21 (76%) liver metastases analyzed. Ten of the 21 (48%) tumors
showed K-ras mutations, 6 of 21 (29%) tumors harbored p53
mutations, and 3 of 21 (14%) tumors had p16 promoter hyper-
methylation (Table 1). The molecular alterations found for each
tumor are summarized in Table 2. Primary screening for muta-
tions in the tumor tissue allowed us to then search for the same
specific mutation in the lymph nodes with a specific mismatch
ligation or MSP assay, both of which are several times more
sensitive (capable of detecting at least 1 tumor cell in 1000
normal cells) than the direct sequencing of PCR products.
Molecular Alterations at the Perihepatic Lymph Nodes
in Colorectal Cancer Patients with Liver Metastases. A
mismatch ligation assay was used to test for K-ras and p53
mutations in the 68 lymph nodes of the 16 patients whose liver
metastases had a mutation, and the MSP assay was used for p16
promoter methylation detection (Fig. 1). Among the 68 lymph
nodes, the examination by standard histopathological proce-
dures diagnosed 17 nodes as a positive (28%), whereas molec-
ular analysis increased this number to 29 (45%). Those lymph
2452 Micrometastases Detection in Colorectal Cancer Patients

Table 2 Comparison of lymph nodes metastasis detection between histopathology and molecular approaches in patients with molecular
alterations in liver tumor DNA
Patient Path. Dxa Molec. Dxa VSa FUa (days) Molecular alteration
a
1 0/2 0/2 D 1280 p53: substitution ATC(195)ACC
2 2/2 2/2 D 163 K-ras: Gly13Asp
3 7/12 8/12 D 6 K-ras: Gly12Asp
p53: substitution AGT(215)AAT
5 0/1 1/1 D 442 K-ras: Gly12Asp
7 0/6 0/6 D 368 K-ras: Gly12Asp
9 2/8 4/8 D 265 K-ras: Gly12Val
11 0/1 0/1 LOFa 4451 K-ras: Gly12Val
12 0/8 2/8 D 37 p53: insertion CAG(284)CAAG
14 0/4 0/4 Aa 8291 p16 methylation
21 0/5 4/5 D 102 p53: substitution CGG(248)TGG
24 0/1 0/1 D 736 p16 methylation
27 0/3 0/3 NAa p53: substitution GGA(266)TGA
28 0/3 0/3 A 10811 K-ras: Gly12Ala
p53: substitution CGG(248)CAG
29 1/6 3/6 D 348 K-ras: Gly12Val
p16 methylation
31 5/5 5/5 D 423 K-ras: Gly12Asp
35 0/1 0/1 A 11601 K-ras: Gly12Val
Any positive 17/68 29/68
a
Path. Dx, pathological diagnosis; Molec. Dx, molecular diagnosis; VS, vital status; FU, follow-up; D, dead; LOF, loss of follow-up; A, alive;
NA, not available.

nodes positive according to the molecular analysis and negative

by H&E staining were confirmed to be negative for cytokeratin
immunostaining. Twelve of the 68 lymph nodes were positive
for tumor cells by molecular evaluation and negative by con-
ventional histopathology, whereas none were positive by his-
topathology and negative by molecular analysis (P 5 0.0005,
McNemar’s test). Using patients as the unit of analysis (rather
than individual lymph nodes), five patients were positive for
both histopathological and molecular evaluation, three patients
were positive by molecular screening but not by H&E staining,
no patients were negative by molecular evaluation but positive
by histopathological study, and eight patients were negative by
both (P 5 0.25, McNemar’s test). Table 2 summarizes these
findings in the lymph nodes and provides the clinical outcome
of each patient. Every lymph node positive for tumor cells by
histopathology was also positive by the molecular assay, indi-
cating a low rate of false negatives inherent to molecular ap-
proaches. As a positive control, we included DNA from the
tumor harboring the specific mutations in each mismatch liga-
tion assay, and, as a negative control, we used DNA without Fig. 1 Evaluation of the presence (1) or absence (2) of neoplastic cells
mutation. Moreover, each K-ras ligation assay included a tumor according to histopathology (H) or molecular analysis (M) in the perihepatic
lymph nodes of colorectal cancer patients with liver metastases. T, tumor
cell line with known K-ras mutations at different dilutions, tissue for each patient. The patient number is indicated to the left of each
confirming a sensitivity of at least 1 tumor cell in 1000 normal panel. A, mismatch ligation assay for K-ras gene mutations. Example of a
cells. More than one molecular alteration in the same tumor was Val (valine) mutation in several lymph nodes from patient 29. A sensitivity
found in three patients. In patient 3, 1 of the 12 lymph nodes was of 1:100 and 1:1000 indicates 1 neoplastic cell in 100 or 1000 normal cells,
respectively. B, mismatch ligation assay for p53 gene mutations. Point
positive for the K-ras mutation but negative for p53. Similarly, mutation in codon 248 resulting in a CGG (arginine) to TGG (serine)
in patient 29, the K-ras assay showed positivity at three lymph change in patient 21. C, MSP to detect promoter hypermethylation at the
nodes, whereas the methylation approach identified only one p16 gene. Lanes U and M indicate the unmethylated and methylated
node as a positive (the same node was positive by histopathol- reaction for each sample. C1 and C2 represent the positive and negative
ogy and markedly positive for K-ras). The discordance is prob- controls for methylation, respectively. At the far right, Lane 2 represents
the negative control (without DNA) for the PCR reaction.
ably due to slight differences in the sensitivity of each assay.
Micrometastasis at the Perihepatic Lymph Nodes and
Survival. We calculated the survival rate in the group of 16 micrometastases at the lymph nodes detected by our molecular
evaluable patients (patients with any molecular alteration in the assays and compared the results with those of a standard eval-
liver tumor tissue) according to the presence or absence of uation by histopathology alone. Based on the molecular data, the

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Fig. 2 Kaplan-Meier analysis
of survival in patients with liver
metastases. A, survival analysis
according to the presence (E;
n 5 8) or absence (M; n 5 7) of
lymph node metastases by mo-
lecular screening (P 5 0.0005).
B, survival analysis according
to the presence (E; n 5 5) or
absence (M; n 5 10) of lymph
node metastases by standard
histopathological evaluation
(P 5 0.011).
median survival for patients with positive lymph nodes was 165
days, whereas the median survival for patients with negative
lymph nodes was 1056 days (P 5 0.0005). The same analysis
based on histopathological evaluation only indicated a median
survival of 175 days for patients with tumor cells at the lymph
nodes and 798 days for patients negative for the same test (P 5
0.011; Fig. 2).
DISCUSSION
The identification of genetic changes associated with neo-
plastic development provides us with a variety of potentially
powerful molecular markers for the clinical detection and fol-
low-up of cancer patients. Tumor-specific molecular abnormal-
ities in several bodily fluids have been investigated as a diag-
nostic tool in many tumor types. Different genetic alterations
have been found in the sputum of lung cancer patients (13), the
stool of colorectal cancer patients (14), the urine of bladder
cancer patients (15), and oral rinses from head and neck cancer
patients (16). In patients with head and neck cancer, molecularly
positive margins provided a high rate of local tumor recurrence
and were correlated with survival (17). Recently, several studies
have been undertaken to evaluate the accuracy and sensitivity of
occult micrometastatic disease detection in the lymph nodes by
molecular-based approaches in many tumor types, including
colorectal cancer (2, 18). Molecular screening of metastases
represents a more sensitive approach compared to cytological
examination and has an additional advantage in that it allows the
examination of the whole lymph node, whereas standard histo-
logical diagnosis only analyzes a few slides. In agreement with
previous reports, our assays for metastasis detection are more
powerful than conventional histopathology (P 5 0.0005, Mc-
Nemar’s test). We recognize that the analysis based on the
lymph nodes assumes that all nodes are independent of one
another. This is not strictly true, because patients contribute
more than one lymph node, making this analysis slightly anti-
conservative. Perhaps most importantly, molecular-based anal-
ysis found positive lymph nodes in three patients classified as
completely negative by histopathological evaluation and cyto-
keratin immunostaining, accounting for the significant differ-
ence in survival outcome based on molecular diagnosis.
The mismatch ligation assay used in the present work has
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Research.
Clinical Cancer Research 2453
been shown to have a sensitivity of about 1 tumor cell in 1000
normal cells, and the MSP assay has a similar sensitivity (9).
MSP evaluation has the advantage of being a a nonradioactive
method and has been found to be reliable in detecting p16
promoter hypermethylation in bronchoalveolar lavages from
lung cancer patients (19). Several approaches for lymph node
micrometastasis detection have been described, and most of
them are based on DNA or RNA isolation followed by PCR.
The DNA-based approaches tended to be absolutely specific
because they detect genetic abnormalities present only in the
neoplastic DNA and not in the normal DNA. Furthermore,
DNA-based techniques can be used robustly in paraffin-embed-
ded tissue, allowing evaluation in retrospective studies. A major
inconvenience for DNA-based tests is that only those tumors
with identified genetic changes can be evaluated. In the present
study, 76% of the patients could be analyzed because they had
at least one molecular abnormality detected by either mutation
analysis or promoter hypermethylation. However, our percent-
age of p16 promoter hypermethylation and p53 mutations was
low compared to those of previous reports (20, 21), probably
due to random sampling. Therefore, a good percentage of pa-
tients evaluable for genetic screening should generally be
higher. Moreover, analysis of other common mutations such as
the APC and b-catenin genes and further elucidation of genetic
changes in colorectal cancer may allow the use of these ap-
proaches to study virtually every patient.
The high sensitivity of the molecular assays can have an
important impact on clinic management. The identification of
metastases in regional lymph nodes is routine practice for sur-
gical pathologists and is one of the most significant prognostic
factors derived from the staging process. In Dukes B colorectal
cancer, the assessment of metastases by molecular techniques in
pathologically negative lymph nodes has been correlated with a
poor prognosis, indicating that molecular-based techniques may
be a useful prognostic factor in colorectal cancer that could also
serve as a selective marker for intensive postoperative adjuvant
chemotherapy (5, 7). Although additional studies with a higher
number of patients must be done to confirm the findings, our
results indicate that the molecular screening of perihepatic
lymph nodes can be a good prognostic indicator of recurrence in
colorectal cancer patients with resectable hepatic metastases. It
2454 Micrometastases Detection in Colorectal Cancer Patients
is likely that the presence of tumor cells at the lymph nodes
indicates secondary spread of the disease from the liver metas-
tases or represents the presence of more widely disseminated
disease from the initial primary tumor. There is little data about
the determinants of prognosis in this specific group of patients.
Some potential prognosis factors include the number of liver
metastases (one to three versus four or more), carcinoembryonic
antigen levels, ploidy, and hepatic lymph node involvement
(22). In agreement with the last parameter, we found that pa-
tients affected with metastases at the perihepatic lymph nodes
have a significantly worse survival than those without lymph
node involvement. Moreover, molecular detection of occult
tumor cells enhances the differences between both groups of
patients. Although the data are still preliminary, the use of
molecular approaches to detect perihepatic lymph node tumor
involvement in colorectal cancer patients with liver metastases
may be a tool in the selection of those patients that can benefit
from liver metastasis resection.
We have used molecular-based techniques (specific mis-
matched ligation assay and p16 promoter hypermethylation) to
increase the sensitivity of tumor cell detection in the perihepatic
lymph nodes from colorectal cancer patients with recurrent
disease. Moreover, molecular detection of occult node metasta-
ses predicts a poor prognosis in colorectal cancer patients with
resectable liver metastases. Prospective studies should evaluate
whether patients positive for tumor cells by molecular analysis
should be subjected to the morbidity of aggressive surgery or
instead entered into trials of intensive adjuvant therapies.
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