(American Journal of Pathology. 2002;160:1001-1008.)
© 2002 American Society for Investigative Pathology
Lectin Histochemistry of Resected Adenocarcinoma of the Lung
Helix pomatia Agglutinin Binding Is an Independent Prognostic Factor
Eckart Laack*,
Haleh Nikbakht*,
Anja Peters
,
Christian Kugler
,
Yvonne Jasiewicz
,
Lutz Edler,
Dieter Kurt Hossfeld* and
Udo Schumacher¶
From the Department of Oncology and Hematology*
and the Institute for Anatomy,¶
UniversityHospital Hamburg-Eppendorf, Hamburg; the Departments ofPathology
andSurgery,
General Hospital Harburg, Harburg;and the German Cancer Research Center,
Heidelberg, Germany
 |
Abstract
|
|---|
The worldwide incidence of adenocarcinoma of the lung is rising.
Unfortunately, no significant prognostic marker beyond the
classical TNM staging exists to stratify these patients for appropriate
therapy. Because lectins, carbohydrate-binding
proteins, have been shown to be useful prognostic
markers in several other adenocarcinomas, a panel
of lectins [Helix pomatia agglutinin (HPA),
Phaseolus vulgaris leukoagglutinin, Ulex
europaeus agglutinin, Maackia amurenis
agglutinin, Sambucus nigra agglutinin] with
different carbohydrate-binding specificities were tested for their
prognostic relevance. Paraffin wax sections of 93 patients with
adenocarcinomas of the lung who had undergone surgery between 1990 and
1995 were investigated by lectin histochemistry. Lectin-binding data
and other known prognostic factors were correlated with survival. In
univariate analysis, binding of HPA, Phaseolus
vulgaris leukoagglutinin, and Ulex
europaeus agglutinin to adenocarcinoma cells were
prognostic indicators for overall and relapse-free survival,
whereas Maackia amurenis agglutinin and Sambucus
nigra agglutinin binding had no prognostic value.
However, in a multivariate analysis next to stage and gender
only HPA was a significant independent prognostic factor on survival.
In conclusion, HPA binding was the primary marker-based
predictor of prognosis in our patient population and allows to stratify
patients with adenocarcinomas of the lung into a low- and a high-risk
group.
In the United States and Western
Europe lung cancer is the most common fatal neoplasm. From a
histological point of view, lung cancer is a heterogeneous group of
tumors, three-quarters being non-small cell lung cancer. In non-small
cell lung cancer surgical resection is the therapy of choice in early
stages of the disease. However, even in this selected group of
patients, approximately half of the patients relapse after complete
resection indicating that the tumor has already spread beyond its
anatomical site at the time of surgery. At present, the prognostic gold
standard is the stratification of patients according to the TMN
classification.1,2
Patients with early disease (stage I
and II disease) have a 5-year survival rate between 30 and 75% after
complete resection. Patients with locally advanced disease (stage
IIIA/B disease) have a 5-year survival rate between 5% and 15%, and
patients with metastatic disease (stage IV disease) have a survival
rate of less than 2%.2,3
Based on their morphology non-small cell lung cancers can be subdivided
into adenocarcinomas, squamous cell carcinomas, and large cell
carcinomas. Adenocarcinomas have been rising in incidence during the
past decades and have become the most common type of non-small cell
lung cancer in Western Europe and in the United States.4,5
Despite the clinical need to stratify adenocarcinomas of the lung with
regard to prognosis beyond the classical TNM classification, no
satisfactory prognostic marker has emerged as yet.
The classical TNM classification rests on the anatomical description of
the tumor spread. The inherent disadvantage of such a classification is
that it is purely anatomical and descriptive and thus does not allow
any functional insight into the metastatic capability of the tumor, the
knowledge of which could lead to innovative therapeutic strategies.
In adenocarcinomas other than lung (eg, breast, colon, stomach, and
prostate) the lectin from the Roman snail, Helix pomatia
agglutinin (HPA), has been used to define the metastatic phenotype of
these tumors.6
Lectins are carbohydrate-binding proteins
and because most of the cellular glycoconjugates are membrane-bound
glycoproteins it has been hypothesized that their altered glycosylation
is functionally involved in the metastatic process thus providing a
rationale for the use of HPA in metastasis research.7
However, HPA has not been the only lectin that has provided useful
prognostic information. The lectin Phaseolus vulgaris
leukoagglutinin (PHA-L) was a useful prognostic marker in breast and
colon cancer8,9
as well as in human diffuse large B-cell
lymphoma.10
A significant correlation between the
expression of PHA-L-binding oligosaccharides and the incidence of the
metastasis to regional lymph nodes in oral squamous cell carcinoma has
also been reported.11
The lectin Ulex europaeus
agglutinin (UEA-I) has been investigated in breast cancer where its
staining was related to disease-free interval and
survival.12
In the study by Fenlon and
colleagues,12
sialidase predigestion was also used before
HPA and UEA-I application. The results indicated that both lectins lost
their prognostic significance indicating that sialic acid can play a
role in masking binding sites for these two lectins. In contrast,
enhanced sialylation of tumor cell glycoproteins has been considered
important for the metastatic capabilities of colorectal carcinomas.
Thus, the sialic acid-specific lectins Sambucus nigra
agglutinin (SNA-I) and Maackia amurenis agglutinin (MAA)
have been discussed as useful prognostic markers in colorectal
carcinoma.13-16
The aim of the present investigation was to analyze the glycoconjugate
expression of adenocarcinomas of the lung with several lectins
differing in their carbohydrate specificity to possibly define a
prognostic marker in this clinically important tumor entity.
|
Patients and Methods
|
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Patients
Tissue blocks containing tumor tissues of 93 patients with
adenocarcinomas of the lung who had undergone surgery between 1990 and
1995 in the General Hospital Harburg, Hamburg, Germany, were
investigated.
Histology and Histochemistry
Formalin-fixed and wax-embedded tissue blocks were used. After
dewaxing, lectin histochemistry was performed using biotinylated
lectins (for lectins, their abbreviation, and sugar specificity see
Table 1
; all lectins were obtained from
Sigma, Deisenhofen, Germany) and an avidin-biotin-alkaline phosphatase
complex was used for visualization (ABC Alkaline Phosphatase; Vector
Laboratories, Peterborough, UK) with a slight hematoxylin
counterstain.17
For HPA only an indirect
immunohistochemical technique [designated as iHPA in contrast to
biotinylated HPA (=bHPA)] was also used because it has been shown that
the staining methodology influences the prognostic
impact.18
For the indirect method, dewaxed sections were treated for 15 minutes
at 37°C with 0.1% trypsin dissolved in lectin buffer. After rinsing
in distilled water, endogenous peroxidase was blocked at room
temperature by incubating the sections in 3%
H2O2 in methanol. After
careful rinses in buffer, the sections were incubated with 10 µg/ml
of HPA for 1 hour at room temperature. After rinsing in lectin buffer,
sections were incubated in normal swine serum (1:5 dilution in buffer)
for 30 minutes. An overnight incubation with a rabbit anti-HPA antibody
(diluted 1:500; EY Lab, San Mateo, CA) followed. After careful rinsing
of the slides, the sections were incubated with a biotinylated
anti-rabbit antibody (1:400; DAKO, Glostrup, Denmark). After rinsing
with buffer, incubation with an avidin-biotin-horseradish peroxidase
complex followed. Diaminobenzidine and
H2O2 were used as
substrates for enzyme visualization.
Sections from the in vitro grown human HT 29 cancer cell
line, which was HPA-positive and metastasized in SCID
mice,19
were used as positive controls. These cells
stained intensively for HPA. Omission of the lectin or preincubation
with 0.3 mol/L N-acetylgalactosamine resulted in an
abolishment of the cancer cell reactivity.
In addition to lectin histochemistry, hematoxylin- and eosin-stained
slides were used for a general overview and to identify area of the
tumors.
The staining of the cancer cells was recorded as follows: negative
indicated no staining or weak staining of single tumor cells (<5%),
positive staining indicated that at least 6% of the tumor cells were
stained (Figure 1)
.20
The
classification into negative or positive was done independently by two
observers who agreed in more than 95% of the cases; in the remaining
cases, consensus was achieved after discussion. The slides were
examined under a Zeiss Axioplan photomicroscope (Carl Zeiss, Jena GmbH,
Jena, Germany) and photographed with a Kodak Ektachrome 64T color film
(Kodak Company, Rochester, NY).
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Figure 1. Biotinylated HPA-positive
(a) and
HPA-negative
(b)
adenocarcinoma of the lung. Original magnification, x200.
|
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Statistical Analysis
All patients were followed-up systematically for a minimum of up
to 5 years. The data of diagnosis, relapse, and deaths were recorded.
From these data survival curves were prepared according to the
Kaplan-Meier method for time to death and time to relapse,
respectively, and were compared with the log-rank
test.21,22
A chi-square test was used to analyze whether
correlations existed between the binding of different lectins and
between each lectin and each categorical clinical factor.
The results of the lectin binding and of the clinical data were
assessed for their predictive value for patient survival by the
proportional hazard model (Cox regression model).23
First
the candidate factors were investigated for their prognostic value in
an univariate analysis using the Cox regression model that is
equivalent to the evaluation of each factor separately using the
log-rank test for differences between prognostic groups determined by
the respective levels of one factor. Variables with sufficient
statistical prognostic power (P < 0.1) were
further analyzed in a multivariate Cox regression model, and
application of a model selection approach to determine a final
parsimonious prognostic model for patient survival based on this data
set. Forward and backward variable selection and the likelihood ratio
statistic were used with the SAS system (SASISTAT Software, Release
6:12; SAS Institute Inc., Cary, NC). The outcome of the Cox regression
was described quantitatively by the statistical estimate of the
regression parameter ß and its SE, its risk ratio exp (ß) and the
respective P value obtained from the Wald test statistic.
Model uncertainty was examined in residual tests and qualitatively
analyzed by the determination of the amount of collinearity in the
respective sets of prognostic variables using pairwise correlation.
|
Results
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Patient Characteristics
The tumor tissues of 93 patients were investigated (for patient
characteristics see Table 2
). The
majority of patients (68%) were male. Patients had a median age of
59 years (range, 27 to 81 years). The tumors of 72 patients (78%) were
pathologically staged as early disease (stages I and II) with no signs
of metastasis in mediastinal lymph nodes, and 19 (20%) were staged as
locally advanced disease with tumor cell-positive lymph nodes of the
ipsilateral mediastinum (stage IIIA). None of the patients showed
tumor-positive lymph nodes of the contralateral mediastinum or distant
metastases. Nevertheless, one patient with a pulmonary metastasis in
the same lobe as the primary tumor (stage IIIB disease) was included as
well as one patient with a pulmonary metastasis in another lobe of the
same side (stage IV disease). Patients with positive mediastinal lymph
nodes received radiotherapy after surgery. The predominant grade of
tumor differentiation was moderately differentiated (40%), followed by
poorly differentiated tumors (35%). The most common blood group type
was A (45%).
Lectin-Binding Characteristics and Survival Analysis
The overall 5-year survival rate of all 93 patients was 49.5%.
Distant metastases or a local relapse were diagnosed in 49 patients
(53%). Tumor stage (log-rank test, P < 0.000005, see
Figure 2
), grade of tumor differentiation
(P = 0.025), and gender
(P = 0.04) had a significant influence on
overall survival. The blood group type did not show any association
with prognosis (P = 0.36).
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Figure 2. Kaplan-Meier plot of overall survival by tumor stage of 93 patients
with resected adenocarcinomas of the lung.
|
|
The tumors of 9 patients (10%) showed no binding of biotin HPA (bHPA),
whereas the tumors of 84 patients (90%) demonstrated moderate to
intense bHPA binding to the tumor cells. Kaplan-Meier survival curves
(Figure 3)
of time to death in months for
bHPA-negative patients versus bHPA-positive patients
revealed a significant difference in survival between the two different
groups (P = 0.015). Six tumors (6%) were
classified as iHPA binding-negative and 87 (94%) as iHPA
binding-positive. The patients with iHPA-negative tumors also had a
significantly longer survival than those with iHPA-positive tumor
tissues (P = 0.025) (Figure 4)
. There was a highly significant
correlation between bHPA and iHPA binding tumors (chi-square test,
P = 0.001).
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Figure 3. Kaplan-Meier plot of overall survival for biotinylated HPA
(bHPA) binding. Patients
(n =
84) whose tumors expressed bHPA-binding sites
had a significantly poorer survival than patients
(n =
9) whose adenocarcinomas were bHPA-negative.
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Figure 4. Kaplan-Meier plot of overall survival for iHPA binding. Patients
(n =
87) whose tumors expressed iHPA-binding sites
had a significantly poorer survival than patients
(n =
6) whose adenocarcinomas were iHPA-negative.
|
|
All patients whose adenocarcinomas were either bHPA- or iHPA-negative
survived 5 years (for patient characteristics see Table 3
). All patients with bHPA-negative
tumors showed no signs of lymph node involvement (N0-status) at the
time of diagnosis. Eight of the nine bHPA-negative patients had a stage
I disease [six patients: stage IA (T1N0M0); two patients: stage IB
(T2N0M0)]. One patient who had a bHPA-negative and iHPA-positive tumor
was staged as IIB disease (T3N0M0). This patient was diagnosed having a
small cell lung cancer with distant metastases as a secondary
malignancy 94 months after resection of the adenocarcinoma of the lung,
and subsequently died 3 months later from this neoplasm.
Factors showing a statistically significant association (chi-square
test) with bHPA-binding pattern (negative versus positive)
are listed in Table 4
.
Sixty-eight patients had a tumor (73%) that reacted with PHA-L. Those
patients who had a PHA-L-positive tumor had a significantly poorer
prognosis than those whose tumors were PHA-L-negative
(P = 0.017, Figure 5
). Seventy-five tumors (81%) were
UEA-I-positive and 29% were UEA-I-negative. Again, this lectin
positivity was also associated with a poorer prognosis
(P = 0.022, see Figure 6
).
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Figure 5. Kaplan-Meier plot of overall survival for PHA-L binding. Patients with
PHA-L-positive tumors (n =
68) had a significantly poorer prognosis than
those whose tumors were PHA-L-negative
(n =
25).
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Figure 6. Kaplan-Meier plot of overall survival for UEA-I binding. Seventy-five
tumors were UEA-I-positive and 18 were UEA-I-negative. The lectin
positivity was associated with a significantly poorer prognosis.
|
|
The predictive value of bHPA, PHA-L, and UEA-I was not limited to the
overall prognosis but was also related to relapse-free intervals.
Patients whose tumors did not bind bHPA, PHA-L, or UEA-I had a
significantly longer relapse-free survival than those patients whose
primary tumors bound these lectins [bHPA (P =
0.015), PHA-L (P = 0.028), UEA-I
(P = 0.039)].
The sialic acid-binding lectins MAA and SNA-I did not have any
significant influence on overall survival [MAA
(P = 0.82), SNA-I (P =
0.68)] or on the relapse-free survival [MAA (P
= 0.86), SNA-I (P = 0.35)].
Prognostic Impact
When tumor stage, grading, age,
gender, blood group, bHPA, PHA-L, UEA-I, MAA, and SNA-I were included
in a Cox regression model, next to stage and gender only bHPA-binding
pattern was identified as a significant independent prognostic factor
in this multivariate analysis (Table 5
and Table 6
, A and B). iHPA was not evaluable
because of nonconvergence of the maximum likelihood statistic of the
Cox regression. Therefore and because of its high correlation with
bHPA, only bHPA was used for further analysis.
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Table 6. Multivariate Cox Regressions Analysis Using Factors with Sufficient
Statistical Power (P <0.1) in Univariate Analysis
and (Part A). Results of a Forward and Backward Variable Selection
Procedure (Analysis of Likelihood) for Those Factors (Part B)
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Patients in the bHPA-negative group showed a considerably better
survival than those in the bHPA-positive group. Because bHPA was the
leading factor in multivariate analysis with a relative risk of
mortality of 8.75, the prognostic power of the two other factors tumor
stage and gender in the bHPA-positive subgroup was further investigated
(Table 7)
. The independent prognostic
power of tumor stage and gender in this subgroup and the risk estimates
were very similar to those in the full set of patient data.
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Table 7. Results of a Forward and Backward Variable Selection Procedure for
Tumor Stage and Gender by only HPA-Binding Positive Patients
(n = 84)
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Discussion
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The present comprehensive lectin-binding analysis of
adenocarcinomas of the lung was undertaken to identify possible new
prognostic markers that could be used to stratify the patients for
adjuvant therapy. Using univariate analysis, we have found that binding
of the lectins HPA, PHA-L, and UEA-I to adenocarcinoma cells of the
primary tumors can be regarded as prognostic indicators for overall and
relapse-free survival after the tumor was surgically removed. In
contrast, the binding of MAA and SNA-I to the primary tumors was of no
prognostic value in our study. However, in multivariate analysis only
HPA binding remained as a significant independent prognostic indicator
having a relative risk of mortality of 8.75, next to stage and gender.
Analysis of only the patients with HPA binding-positive tumors,
revealed an independent prognostic power of stage and gender in this
group of patients with very similar estimates to those obtained in the
full set of patient data. Patients who had a HPA-negative,
well-differentiated tumor, and tumor stage IA disease had the best
prognosis. All patients with HPA-negative tumors did not show signs of
lymph node involvement (N0-status) at the time of diagnosis. These
findings are similar to those in breast cancer in which a strong
association between HPA staining in primary tumor and the presence of
metastases in the local draining lymph nodes was
observed.24
However, only 10% of patients had a HPA-negative tumor, which
demonstrates the high malignant potential of adenocarcinomas of the
lung and this observation is concurrent with the high rate of relapses
in our study group. From this we conclude that for our patient
population it is reasonable to use HPA as the primary predictor of
prognosis and that in the larger part of the HPA-positive population
the factors stage and gender represent further important prognostic
factors. However, these findings should be validated in further
studies.
Our results are comparable to those that showed an association between
HPA binding and prognosis in breast, colon, and gastric
cancer,6,7
whereas no prognostic significance for HPA
could be detected in squamous cell carcinoma of the head and
neck.25
Taking the results of these studies together it
becomes apparent that HPA is particularly well suited to recognize a
glycotope on the histological entity adenocarcinomas, where it is equal
or even superior to other classical markers of prognosis. The
metastatic potential of HPA-positive tumor cells has also been
confirmed in xenograft models of breast and colon cancer. When
transplanted into severe combined immunodeficient (SCID) mice,
HPA-positive human breast and colon cancer cells metastasized whereas
HPA-negative cancer cell lines in general did not.19
The molecular basis why HPA binds preferentially to metastasizing
adenocarcinomas has not been elucidated completely. HPA has a
specificity for N-acetylgalactosamine (GalNac) and as this
carbohydrate is part of the blood group A carbohydrate determinant, one
could speculate that it is the blood group A determinant that is
recognized by HPA. However, this is not the case in our study as 84%
of the tumors showed HPA binding whereas only 45% of the patients were
of blood group A indicating that GalNac containing glycoconjugates
other than blood group A substance are responsible for HPA positivity
in the adenocarcinoma cells. This apparent discrepancy between HPA
binding to the cancer cells and the blood group of the patients was
also noted in breast cancers. The monoclonal antibody BRIC 66 directed
against blood group A substance and HPA were both used on tissue
sections and on Western blots of extracted breast cancer glycoproteins.
The results indicated that HPA recognizes blood group A substance if it
is expressed by the cancer cells, but its binding pattern is much
broader and more heterogeneous than that of the monoclonal anti-A
antibody.26
The precise structure of the HPA binding oligosaccharide associated
with metastasis has not been identified yet, however, a monosialylated
oligosaccharide of 4.58 glucose units termed HPAgly-1 has been
identified in HPA-positive breast cancers, whose functional role has to
be elucidated in future studies.27
Two different methods were used to study HPA binding to primary tumor
cells. This was undertaken as much controversy had arisen concerning
the predictive value of HPA in metastasis research in the early years
of its usage.28,29
In the original contribution by Leathem
and Brooks,20
HPA-binding sites were detected by an
indirect method. The lectin was applied to the tissue section and
subsequently localized by an anti-lectin antibody and an anti-antibody
peroxidase anti-peroxidase complex (PAP-technique). Using this
methodology, a significant difference in survival was seen between the
HPA-positive and -negative patients. In contrast, Gusterson and
colleagues29
who were unable to see any prognostic
significance for HPA binding, used HPA covalently linked to horseradish
peroxidase (direct method). This discrepancy in the results was later
resolved by Brooks and colleagues,18
who used both
techniques in one patient cohort. Again, only the indirect method gave
good results indicating that the technique for HPA binding is crucial.
In the present study of adenocarcinomas of the lung, a slightly
modified indirect technique (=bHPA) was used. Here, HPA was linked to
the small molecule biotin, which was subsequently detected by an
avidin-alkaline phosphatase complex. Using this method, Thies and
colleagues30
could show that biotinylated HPA (=bHPA) was
able to identify those malignant melanomas, that metastasized. Because
the indirect and the biotinylated HPA method were both suitable to
identify the metastasizing tumors,30
we wanted to compare
the relative merits of both methods in adenocarcinomas of the lung.
However, the staining results of both methods do not dramatically
differ in this study. From our results we cannot conclude that
bHPA-negative patients differ in their prognosis from iHPA-negative
patients. Therefore further investigations into the prognostic power of
both methods are needed at least in adenocarcinomas of the lung. In
this tumor entity it seems likely that a large number of patients needs
to be examined before one would be able to find differences between
bHPA and iHPA as negative cases seem to occur at a rate of less than
10%.
In summary, using the appropriate methodology, HPA binding to the cells
of primary adenocarcinomas of the lung is a significant independent
prognostic factor. Our results add weight to the importance of HPA as a
prognostic marker and could be an important further step toward the
stratification of patients with adenocarcinomas of the lung into low-
and high-risk populations.
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Footnotes
|
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Address reprint requests to Eckart Laack, M.D., Internal Medicine, Department of Oncology and Hematology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, D-20246, Hamburg, Germany. E-mail: laack{at}uke.uni-hamburg.de
Supported by the Gustav Spierling Stiftung Hamburg, Germany.
Accepted for publication December 7, 2001.
|
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