Prediction of Occult Neck Metastases in Laryngeal ... · pendent predictors of occult nodal disease in laryngeal squamous cell carcinoma, and their immunohistochemical determination
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Vol. 2, 1801-1808, October 1996 Clinical Cancer Research 1801
Prediction of Occult Neck Metastases in Laryngeal Carcinoma: Role
of Proliferating Cell Nuclear Antigen, MIB-1, and E-Cadherin
Immunohistochemical Determination
Alessandro Franchi, Oreste Gallo, Vieri Boddi,
and Marco Santuccit
Institute of Anatomic Pathology [A. F., M. S.]. Otolaryngology Clinic[0. 0.], and Institute of General Pathology [V. B.], University of
Florence, Viale 0. B. Morgagni 85, 50134 Florence, Italy
ABSTRACT
The aim of this study is to investigate the predictive
value of proliferative activity assessment and E-cadherin
expression by means of immunohistochemistry in identify-
ing patients with laryngeal squamous cell carcinoma at a
high risk for occult node metastasis. Thirty consecutive
patients treated for laryngeal carcinoma with false clinically
negative nodes (occult metastases, pN+) between the years
1980 and 1990 were selected for this study. A group of 30
cases with negative cervical lymph nodes (pN-) having a
similar anatomic site and tumor size distribution was used
as control. In each case, several histological parameters,
including grade, pattern of invasion, number of mitosis (X 10
high-power field), tumor inflammatory infiltrate, and tumor
sclerosis, were assessed. Proliferative activity was deter-
mined using immunohistochemical staining for proliferating
cell nuclear antigen (PCNA) and MIB-1. Other putative
prognostic factors investigated at the immunohistochemical
level were the cell adhesion molecule E-cadherin and two
oncoproteins, p53 and c-erbB-2. In pN+ cases, the expres-
sion of PCNA and MIB-l was significantly higher than in
the pN- group. Moreover, a significant loss of E-cadherin
expression was observed in carcinomas with occult metas-
tases. No differences in p53 and c-erbB-2 oncoproteins were
found between pN+ and pN- cases. Among the other path-
ological parameters examined, only histological grade was
significantly associated with the presence of occult metasta-
ses, but on mubtivariate analysis, this relationship was lost.
We conclude that PCNA, MIB-1, and E-cadherin are inde-
pendent predictors of occult nodal disease in laryngeal
squamous cell carcinoma, and their immunohistochemical
determination could be useful in identifying patients with
clinically negative lymph nodes who are at considerable risk
for occult metastases and who may benefit from elective
neck dissection.
Received 3/26/96: revised 7/3/96; accepted 7/3/96.I To whom requests for reprints should be addressed. Phone: 39 55416951; Fax: 39 55 4379868.
INTRODUCTION
Preoperative identification of occult lymph node metastasis
represents a crucial point in the clinical management of patients
with laryngeal cancer for its prognostic and therapeutic impli-
cations (I). Data from literature indicate that 4-40% of patients
with laryngeal carcinoma and clinically negative neck lymph
nodes have indeed occult metastases on subsequent histological
examination (2-6). As a consequence, treatment of N0 patients
is a matter of great controversy between those who are in favor
of a “wait and see” attitude and those who prefer elective neck
dissection. Indeed, prophylactic neck dissection eliminates the
risk of late metastases in N0 cases, but exposes the patient to a
higher morbidity and higher hospital costs, which are in most
cases unnecessary ( I , 3). Another possible disadvantage of
bymphadenectomy may be the local reduction of the immuno-
logical host defense, which may facilitate the spread of residual
tumor cells. Therefore, the identification of biological tumor
factors indicative of high or low risk of occult lymph node
metastases would be extremely helpful in deciding on elective
or delayed therapeutic strategy in the treatment of the neck in N0
laryngeal cancer.
Previous clinicopathobogical studies have shown that the
presence of locoregional metastases in patients with laryngeal
carcinoma is significantly associated with some histological
features of the primary tumor, such as the degree of differenti-
ation, tumor growth pattern, presence of peritumoral desmopla-
sia, and inflammation (7-9). Additional studies confirmed that
histological grade and host immune response correlate signifi-
cantly with occult lymph node metastasis (1 , 10). However,
because of the absence of established criteria, the evaluation of
this group of histological parameters is very subjective, and
reproducibility is poor.
In recent years, the efforts of several research groups have
bed to a dramatic improvement in the understanding of the
neoplastic growth process and have furnished novel molecular
tools that may help in identifying tumors more likely to metas-
tasize, being therefore potentially useful for management and
prognostic purposes. In particular, several lines of evidence
suggest that the evaluation of tumor cell proliferative activity,
cell adhesion markers, and oncoprotein expression could allow
an estimate of the biological behavior of head and neck squa-
mous cell carcinoma. When proliferative activity was evaluated
by tumor labeling index in a series of head and neck squamous
cell carcinomas, the results indicated that lesions with a high
proliferative activity have a poorer survival (1 1). Recently,
Benazzo et a!. observed a significant correlation between pro-
liferative indices measured after in vivo bromodeoxyuridine
incorporation and tumor dimension, histological differentation,
and lymph node involvement in a group of patients with carci-
nomas of the head and neck ( 1 2). Similar results were obtained
1802 Predictors of Occult Metastases in Laryngeal Carcinoma
2 The abbreviations used are: PCNA, proliferating cell nuclear antigen;
OR, odds ratio; Cl, confidence interval.
Table 1 C linicab features of 60 pa tients with 1arynge al squam ous cell carcinoma, the obj ect of this study
Site T” Treatment
Partial Total Age (Mean) Sex M:FSupraglottic Glottic 1 2 3 4
pN+ 20 10 7 8 10 5 13 17 58.5 29:1
pN- 20 10 8 9 8 5 16 14 59.3 29:1
a TNM staging.
when proliferation was measured by flow cytometric analysis of
DNA content (13).
Loss of intercellular adhesion might be an important event
in the early phases of the metastatic process. The potential
relationship between the expression of the adhesion molecule
E-cadherin, a member of a family of calcium-dependent cell-cell
adhesion molecules also including P- and N-cadherin, and thepresence of lymph node metastasis in laryngeal carcinoma has
been the object of studies by different groups. Schipper ci’ a!.
observed that E-cadherin expression decreased with loss of
differentintion in primary cnrcinomns, and thnt lymph node
metastases expressed a lower bevel of the protein. suggesting an
important role of cadhenn loss in the metastatie process ( 14). In
a subsequent study, Mattijssen e� a!, were unable to find anysignificant relationship between the level of li-eadherin expres2sion and the presence of lymph node metastasis in a group of 50
head and neck squamous cell carcinomas ( I 5) However. these
studies only examined patients with clinical evidence of mela-
static disease. and no information is available in the literature
about the relationship between E-cadherin expression and the
presence of occult lymph node metastases,Indeed, most of the studies concerning predictors of the
behavior of carcinomas of the head and neck have consideredheterogeneous groups of lesions concerning both the site of
primarylesion and the clinical node status, The present study is
focused on a homogeneous population of patients with laryngealsquamous cell carcinoma and clinically negative neck lymph
nodes with the aim of defining prognostic parameters that per�mit the identification of patients at risk for occult metastases,We evaluated several histopathological features of primary le-
sions, and determined immunohistochemically tumor cell pro-
liferative activity and E-cadherin expressionS Moreover, westudied the expression of e-erbB-2 and p53 oneoproteins. the
products of two genes known to be involved in the development
of head and neck squamous cell carcinomas (16-18),
PATIENTS AND METHODS
Cases Studied. Thirty consecutive patients treated at the
Institute of Otorhinolaryngology of the University of Florence
between 1980 and 1990 for laryngeal squamous cell carcinomawith occult baterocervical metastases were selected for thisstudy, All patients had clinically negative laterocervical lymphnodes and underwent elective neck dissection, with subsequenthistological recognition of one or more occult metastases
(pN + )‘ A group of 30 patients treated in the same period for
laryngeal squamous cell carcinoma with clinically and histobog-
ically negative laterocervical lymph nodes (pN-) was used as
control, Clinical data of the two groups of patients are summa-ri�ed in Table 1.
Formalin-fixed, paraffin-embedded tissue samples from re-
section specimens were retrieved in each case, and serial see-
tions were stained with H&E or used for the immunohistochem-
ical studies. In each case, the following histological parameters
were assessed: histological degree of differentiation (well, mod-
erately, or poorly differentiated), pattern of invasion (pushing or
infiltrating), number of mitoses (X 10 high-power field), peritu-
moral inflammatory infiltrate (present or absent), and peritu-
moral sclerosis (present or absent).
Immunohlstochemlstry. lmmunohistochemical studies
were performed by the uvidin-biotin complex technique (Duko-
putts. High Wycombe. UK)� Proliferative activity was investi-
gated using monocbonul antibodies against Ki-67 (MIB-l. lm-
munotech, Marseille, France) and PCNA2 (PC-lO, DAKO
Corp,, Carpinteria, CA). For E-cudherin detection, the mono-
clonal antibody HECD- 1 (Nuclear Laser Medicine, Milan, Italy)was employed, For p53 and c-erhB-2 detection, the monoclonal
antibodies D07 (DAKO Corp.) and mAhI (Triton Diagnostics.
Alameda, CA) were used, MIB-1, E-cadherin, and p53 immu-
nostuining required microwave pretreatment of dewuxed and
rehydrated sections, Negative controls were performed by sub-stitution of the primary antibody with nonimmune rabbit ormouse serum,
Scoring of the immunostaining was performed by oneobserver (A, F.). who had no knowledge of patients’ clinical
status, using a standard light microscope equipped with an
eyepiece grid of 10 X 10 squares and a X40 objective, In each
case, at beast 1000 tumor cells were counted from areas withhigh and low expression of the antigen. All stained cells were
considered positive regardless of the intensity of the staining.
and the results were expressed in percentage of’ positive cells.
An arbitrary cutoff value of 20% positive cells was chosen to
separate cases with high and low expression of the antigens.
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TX) The relationship between clinical and pathological van-
ables was assayed by Fisher’s exact test, The correlation be-
tween the different immunohistochemical indices was evaluated
using Spearman’s correlation coefficient. The role of each pos-sible prognostic factor (univaniate analysis) and the joint effect
of all these factors (multivaniate analysis) was explored using
the multivaniate logistic regression analysis, The final results of
these analysis are the ORs and their 95% CIs, The likelihood
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