I. Introduction
Cervical cancer is the most common cancer,
which causes a significant health problem in Thai women. Approximately 5,500
Thai women develop invasive cervical cancer per year (Deerasamee et al, 1999).
Among these, 15-25% are in early stage of disease (stage Ib or IIa)
(Manusirivithaya, 2001a). These patients are usually treated by radical
hysterectomy and pelvic lymphadenectomy, yielding a 5-year survival rate of
80-90% (Landoni et al, 1997; Kim et al, 2000).
Among various prognostic factors, lymph
node metastasis is the most significant predictor of survival, and also
influences on the rate of recurrence (Burke et al, 1987; Larson et al, 1988;
Wilailak et al, 1993; Manusirivithaya et al, 2001b). Its presence necessitates
postoperative adjuvant therapy. Despite the adjuvant treatment, recurrences of
disease in patients with positive nodes are found as high as 33-42% (Burke et
al, 1987; Larson et al, 1988; Manusirivithaya et al, 2001b). However, patients
without pelvic lymph node metastases are not absolutely free from tumor
recurrences, albeit at lower rate, it still occurs in 7-11% (Burke et al, 1987;
Larson et al, 1988; Manusirivithaya et al, 2001b). One possible explanation of this finding
is the cancer cells might have already been existing in these negative pelvic
nodes, but are not recognized from the routine Hematoxylin-Eosin staining (H/E
staining).
In recent years, the advantage of
immunohistochemical techniques using anticytokeratin antibodies has been
demonstrated in identification of micrometastases in histologically negative
lymph node on routine H/E staining. Multiple types of cancer have been reported
with the positive result of this technique, such as breast cancer (Trojani et
al, 1987; McGuckin et al 1996; Reed et al, 2004), colorectal cancer (Cutait et al, 1991; Greenson et al, 1994; Broll
et al, 1997; Oberg et al, 1998; Isaka et al, 1999; Yasuda et al, 2001), gastric cancer (Fukagawa et al, 2001; Nakajo et al, 2001; Siewert
et al, 1996), esophageal cancer (Natsugoe et al, 1998; Glickman et al, 1999;
Komukai et al, 2000; Matsumoto et al, 2000; Mueller et al, 2000; Sato et al,
2001), gall bladder
cancer (Yokoyama et al, 1999; Nagakura et al, 2001), lung cancer (Osaki et al, 2002), endometrial
cancer (Yabushita et al, 2001; Bosquet et al 2003), prostate cancer (Wilcox et
al, 1998), thyroid cancer (Qubain et al, 2002), oral and
oropharyngeal cancer (Stoecki et al, 2002). However, the benefit of
immunohistochemical staining in detection of occult lymph node metastasis is
not consistent in all studies. One study in vulvar cancer did not find any
positive node from immunohistochemical staining in addition to H/E staining
(Leys et al, 2000).
To our knowledge, there have been few
studies reported on occult positive node by immunohistochemical staining in
cervical cancer (Auger and Cogan, 1990; Juretzka et al, 2004; Lentz et al,
2004). The objective of our study was to examine the rate of occult
node positive detected by immunohistochemical staining in cervical cancer
patients, who had undergone radical hysterectomy with pelvic lymphadenectomy,
and had negative nodes from H/E staining, who eventually developed tumor
recurrence.
II. Materials and Methods
A. Patients
The medical
records including personal data, tumor characteristics and follow-up
information of all stage Ib and IIa cervical cancer patients, who were treated
by radical hysterectomy with pelvic lymph node dissection between January 1992
and December 1998, in Maharaj Nakorn Chiangmai Hospital and Bangkok
Metropolitan Administration (BMA) Medical College and Vajira Hospital were
reviewed. The inclusion criteria were: 1) patients with tumor histology of
squamous cell carcinoma, adenocarcinoma, or adenosquamous cell carcinoma 2)
patients who had negative lymph node reported from H/E staining 3) developed tumor recurrence. The
exclusion criteria were: 1) past or present history of other cancers (two
primary cancers) 2) unavailable
paraffin blocks of lymph nodes.
B. Immunohistochemistry study
Paraffin–embedded
tissue blocks of all dissected nodes from the eligible patients were
identified. Single section of 3-micrometer in thickness was cut from each
paraffin block. Paraffin sections were dewaxed with xylene and treated with 95%
alcohol. Sections were then treated with 3%H2O2 in
phosphate buffer saline (PBS) to block endogenous peroxidase activity. For
antigen retrieval, they were immersed in 10 mM/L citrate buffer (pH 6.0), and
microwaved at 750 W power for 10 minutes. After the buffer had cooled, sections
were treated with anticytokeratin antibody (AE1/AE3) diluted 1:300 for 60
minutes at room temperature. Sections were treated for another 10 minutes with
the biotinylated link antibody (Dako LSAB code no. K0675 bottle 1). After being
rinsed in PBS, the sections were coated with streptavidin-HRP (Dako LSAB code no.
K0675 bottle 2) for 10 minutes. The reaction product was developed with
diaminobenzidine solution for 10 minutes. Sections were then counterstained
with Harry hematoxylin, dehydrated through 95% alcohol and absolute alcohol,
and were mounted. Primary cervical cancer specimen was used as positive
control. The negative control consisted of sections that were treated with the
same technique with the primary antibody omitted.
Occult node
positive or micrometastasis was defined as a single tumor cell or cluster of
tumor cells that were not evidenced on conventionally H/E staining but were
detected by anticytokeratin immunohistochemical staining.
III. Results
During the study period, 39 recurrent
cervical cancer patients met all other eligible criteria. Five cases with
unavailable pathological paraffin blocks of lymph nodes were excluded. Totals
of 34 patients with 1,012 nodes were included in the study. The median age of
the patients at the time of surgery was 38 years (range, 33-67 years). Other
clinical and pathological characteristics of the patients and tumors are
demonstrated in Table 1. Approximately 94% of the patients
were in stage Ib. Approximately 62% of the tumors were squamous cell carcinoma;
and at the same percentages, the tumor were grade 2. Lymph-vascular space
invasion were present in 68%, and depth of invasion were more than half of
cervical thickness in 77%. The primary tumor size ranged from no definite gross
lesion to 6 cm in maximal diameter with the median of 2.7 cm.
Local recurrences occurred in 23 patients
(68%), five patients (15%) had isolated distant recurrence, and six patients
(17%) had both local and distant recurrences. The recurrence-free interval from
primary surgery ranged from 5-67 months with the median of 15 months. About 70%
of the recurrence were evidenced within 2 years after surgery, and 94% were
diagnosed within 5 years. The treatment for tumor recurrences were radiation
alone in 15 cases (44%), radiation and chemotherapy in seven cases (20%), and
chemotherapy alone in two cases (6%). Two patients underwent surgery followed
by chemotherapy alone or chemotherapy and radiotherapy in each of them. Eight
patients declined any treatment. By the time of this report, 18 patients (52%)
were dead, eight (24%) were lost to follow up, and eight (24%) were doing well
without evidence of disease at their last follow-up visit. The overall 5-year
survival rate after recurrence was 44.0% (95%CI: 18.3%, 62.3%).
From the total number of 1,012 negative
lymph nodes from 34 patients, most were from the obturator group as 424 nodes
(42%); the others were external iliac, common iliac and paraaortic nodes as 304
nodes (30%), 236 nodes (23%), and 48 nodes (5%) respectively. The number of
nodes evaluated in each patient ranged from 6-70 nodes with the median of 26
nodes. Approximately 74% of patients had more than 20 lymph nodes dissected
from each of them. All of these negative nodes from H/E staining were also
negative for cytokeratin from AE1/AE3 immunohistochemical staining.
polykeratin antibody was unlikely to increase the sensitivity in
detection of lymph node metastases in cervical cancer. The other study of
Juretzka et al (2004) attempted to explore further on the association between
the clinical outcome and the positive staining. Their study used the AE1/ CAM
5.2 immunohistochemical staining to detect the occult node micrometastasis, not
initially identified by H/E staining in 976 nodes from 49 stage Ia2-Ib2
cervical cancer patients who underwent radical hysterectomy. Two sections from
each paraffin tissue block were immunohistologically studied. They could
identify micrometastasis in only four nodes from four patients. All of these
four patients had other poor prognostic features including lymph-vascular space
invasion (n=3), >4 cm in size of primary cancer (n=2). With the mean follow
up of 39 months, higher proportion of patients with micrometastasis had recurrences
compared to those with negative micrometastasis (2 of 4 patients versus 3 of 45
patients respectively). However, the numbers of patients in this study was too
small to draw any conclusion regarding the clinical implications of the
immunohistochemically occult node metastasis in cervical cancer.
Recently, Lentz et al (2004) studied the
prevalence of micrometastasis in 3,106 histologically (H/E staining) negative
lymph nodes from 132 cervical cancer patients who were in stage Ia-Ib2.
Micrometastases were present in only 29 nodes (1%) from 19 patients (15%
[95%CI; 9%, 22%]). The patients who had > 20 resected nodes had
higher detection rate than those with <20 nodes (26% compared to 2%). Since
the rate of the patients with micrometastatic lymph node (15%) was remarkably
approximate to the rate of patients who eventually experience recurrence in the
apparently negative node patients, the authors proposed that these patients
with micrometastatic node should be the same group of patients who would experience
recurrence. However, their study did not provide any follow up information, so
the hypothesis remained to be proven. If the hypothesis of Lentz et al is
valid, immunohistochemical staining with anticytokeratin antibody should lead
us to a more precise identification of the patients who are at risk of
recurrence, and this particular group of patients would certainly gain benefit
from the adjuvant postoperative treatment.
Our study was limited to stage Ib to IIa
cervical cancer patients, who had negative node by conventional H/E staining
but eventually developed tumor recurrence. We expected that these patients
would probably have high rate of occult node positive. Unexpectedly, we could
not demonstrate any occult positive node in all 1,012 nodes from the 34
recurrent patients. Our result was different from the previous reports
(Juretzka et al, 2004; Lentz et al, 2004). Juretzka et al, (2004) although
found only 1% prevalence of occult node positive but they found obviously
higher recurrences in the occult node positive patients compared to those with
occult node negative, 50% versus 7% respectively. While Lentz et al, (2004)
reported the prevalence of micrometastasis as 15% in stage Ia2-Ib2 cervical
cancer irrespective of recurrence. We can hardly postulate the total negative
occult node in our study despite the high-risk condition of the patients. All
of our patients had tumor recurrence; most had other poor prognostic factors as
presence of lymphovascular space invasion (LVSI) (68%) and deep tumor invasion
(76%). Regarding the number of lymph nodes retrieved from each patient, which
was found to be a significant factor associated with the rate of occult node
metastasis (Isaka et al, 1999; Matsumoto et al, 2000; Lentz et al, 2004),
approximately 74% of our patients had more than 20 nodes harvested.
The limitation of our study was the only
single number of section from each lymph node block was evaluated by
immunohistochemical staining. Theoretically, in order to detect all possible
metastases, all nodes should be serially cut and stained. However, this process
could not be applied in general practice because it is impractical, labor
intense, and costly. Nevertheless, this factor may not be the rationale to our
negative result because the studies which demonstrated the prognostic
significance of occult node positive in other cancers also evaluated only
single section of each block of lymph node (Greenson et al, 1994; Siewert et
al, 1996; Natsugoe et al, 1998; Yokoyama et al, 1999; Mueller et al, 2000;
Nagakura et al, 2001; Nakajo et al, 2001; Yabushita et al, 2001). Even the
study of McGuckin et al, who conducted their study with four-level
sections did not find a significant increase of the positive detection rate for
cytokeratin immunohistochemical staining over one-level section (McGuckin et
al, 1996).
The other difference between our study and
the study of Juretzka et al, (2004) and Lentz et al, (2004) is the type of
primary antibody for cytokeratin. We used AE1/AE3 antibody while they used
AE1/CAM 5.2 Antibody (Juretzka et al, 2004; Lentz et al, 2004). AE1/AE3 is a
mixture of two antibodies (AE1 and AE3), which react to a broad spectrum of
human keratins. AE1 reacts to most acidic keratin (type I), while AE3 reacts
with most basic (type II) cytokeratin (Moll et al, 1982). CAM 5.2 is a mouse
monoclonal antibody specialized for cytokeratin 8 and 18 cytokeratin (Moll et
al, 1982). We select AE1/AE3 for this study based on the result from
FukagawaÕs study, which found that AE1/AE3 is the most sensitive antibody for
the detection of micrometastasis compared to KL-1 and CAM 5.2 (Fukagawa et al,
2001). However, this statement might be questioned from the negative result of
our study whether it is a truly sensitive antibody. On the other hand, the
negative result of our study would be affirmed.
One could question that the aging of the
paraffin blocks might be the reason for negative micrometastasis node in our
study. However, both the primary tumor and the positive node of other patients
who were operated during the same period clearly demonstrated positive
cytokeratin immunohistochemical staining. Hence, the aging of the blocks was
unlikely to be the reason for our negative result. We studied the tissue blocks
from patients who were operated during the period of 1992 to1998 because this
retrospective descriptive study was destined to be the first phase trial. The
forthcoming case-control study was intended, if the immunohistochemical
staining for cytokeratin could demonstrate a high incidence of occult node
metastasis in patients with tumor recurrence. In this circumstance, the
controls of the planned study should be those who are free of recurrence for at
least 5 years after surgery.
In
conclusion, in this study, immunohistochemical stiaining with AE1/AE3 antibody did not have any advantage
over the conventional staining in demonstration of an occult positive node in
any of the early stage cervical cancer patients, even in the high-risk group of
patients who eventually developed tumor recurrences.
Acknowledgements
The authors would like to thank Ms. Aree
Pantusart for her kindly help in gathering the clinical data, Ms. Lakana
Eienleng for doing all the immunohistochemical stainings, the staffs in the
Department of Pathology of both hospitals for their co-operation. The authors
also appreciate the Medical Research Fund of the BMA Medical College and Vajira
Hospital for the grant support of this research.
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