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Peritoneal Cytology Does Not Increasethe Prognostic Information Provided by TNMin Gastric Cancer
G. de Manzoni, MD,1 G. Verlato, MD,2 A. Di Leo, MD,1 A. Tomezzoli, MD,3
C. Pedrazzani, MD,1 F. Pasini, MD,4 Q. Piubello, MD,3 C. Cordiano, MD1
1First Division of General Surgery, University of Verona, 37126 Verona, Italy2Unit of Epidemiology and Medical Statistics, University of Verona, 37126 Verona, Italy3
Division of Pathology, Borgo Trento Hospital, Verona, 37126 Verona, Italy4Division of Medical Oncology, University of Verona, 37126 Verona, Italy
Abstract
Background: This study aimed at verifying whether peritoneal cytology could improve the prog-
nostic information provided by TNM staging in gastric cancer patients.
Method: The presence of free peritoneal tumor cells was investigated in 168 patients who
underwent curative resection for gastric cancer from January 1992 to July 2002 in Verona, Italy.
The influence of peritoneal cytology on survival was evaluated by a Cox regression model, con-
trolling for potential confounders.
Results: Twenty-three patients (14%) had positive peritoneal cytology. Patients with positive la-vage were more likely to present serosal infiltration (100 vs. 46%) and nodal metastases (91 vs.
67%; P < 0.001). Positive lavage was associated with a very poor prognosis: 3-year survival was
only 9% (95% CI 227%) when peritoneal cancer cells had been detected, whereas survival
reached 50% (95% CI 4259%) in patients with a negative cytology. In multivariate survival
analysis, peritoneal cytology was an independent predictor of mortality when controlling for sex,
age, site, histology, and nodal metastases, but not when adjusting also for depth of tumor invasion
(RR of positive versus negative = 1.2, 95% CI 0.72.0). Similarly, the influence of peritoneal
cytology on survival was no longer significant when univariate analysis was restricted to T3/T4
patients (RR = 1.5, 0.92.5).
Conclusions: Positive peritoneal cytology was a marker of poor prognosis in gastric cancer pa-
tients. Nevertheless, peritoneal lavage did not increase the prognostic information already pro-
vided by the TNM staging system in this Italian series.
P eritoneal metastases are the most frequent type ofrecurrence in patients with gastric cancer, evenafter potentially curative resection, especially for diffuse
neoplasms with serosal invasion.1 The causal relation-
ship between infiltration of serosal layer, dissemination of
malignant cells, and their peritoneal implantation has
been well described, and free cancer cells identified by
cytology of intraoperative peritoneal lavage could be a
strong indicator of future peritoneal metastases.24
However, many patients with negative cytology will de-
velop peritoneal metastases, and some authors deny that
cytological findings in peritoneal lavage can be useful in
predicting peritoneal recurrence.5
This study aimed at verifying whether peritoneal cytol-
ogy could improve the prognostic information provided byCorrespondence to: G. de Manzoni, MD, e-mail: nadaffona@inter-
free.it; [email protected]
2006 by the Societe Internationale de Chirurgie World J Surg (2006) 30: 579584
Published Online: 10 March 2006 DOI: 10.1007/s00268-005-7901-2
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the TNM staging in gastric cancer patients, with respect
to peritoneal recurrence and disease-related survival.
METHODS
Patients
Intraoperative cytological examination was carried out
before dealing with tumors in 196 patients who underwent
gastrectomy for gastric cancer in the First Department of
General Surgery, University of Verona, from 1 January
1992 to July 2002. These patients represented 55% of all
the patients operated on for the disease in that period
(n = 359). After excluding 8 patients whose lavage was
classified as inadequate, 16 patients who underwent R2
resections or presented microscopic residual disease at
the resection margins, and 4 patients who died of post-
operative complications, 168 patients were recruited for
the study. In all these patients at least D2 lymphaden-
ectomy had been performed.
Tumors were classified according to the 1997 patho-
logic classification (pTNM) of the International Union
Against Cancer,6 and histological classification followed
the criteria of Lauren.7
Peritoneal Lavage Cytology
Immediately after the abdominal cavity was opened,
200 ml of physiological saline was introduced in the in-
framesocolic region, and after manually stimulated dis-
persion of the saline, a sample of about 50 ml was
recovered from the Douglas pouch.
In all the 168 cases, the collected samples underwent
centrifugation (2500 rpm per 5 minutes), and four slides
of samples were prepared for extemporary cytological
examination, always carried out by the same expert
cytologist (A.T.) through rapid fixing with alcohol 95% and
rapid coloration with haematoxylin-eosin. In cases of
negative extemporary cytological examination, two moreslides were prepared with the same procedure regarding
centrifugation and cytocentrifugation, but using the Pa-
panicolau stain for the final examination. Each slide was
classified as inadequate, negative, or positive. Slide
preparations were considered inadequate if there was
insufficient cellular material for a diagnosis, if blood col-
oration was so heavy that it prevented cellular examina-
tion, or if there was a fault in fixation or coloration.
The cytological samples already prepared with stan-
dard coloration were re-examined with immunocyto-
chemistry by another expert cytologist (Q.P.), who was
unaware of the previous cytological response. Two dif-
ferent antibodies were used: one for epithelial cells (Ber-
EP4 1:50) and one for mesothelial cells (anti-Calretinin
1:300) as negative control.
Follow-up
All patients, after being discharged from hospital, were
scheduled for outpatient follow-up examinations every 6
months. None of the patients was treated with perioper-
ative or postoperative chemotherapy. Follow-up was
completed in June 2005. Patients classified as disease-
free at the last follow-up examination presented com-
pletely normal results from all studies of images as well
as normal tumor marker levels. The median follow-up for
surviving patients was 64 (range: 35159) months.
Statistical Analysis
Significance of differences between patients with and
without free cancer cells after the cytological examination
of the peritoneal lavage were assessed by a t-test for
continuous variables (age), by the v2 test or Fisher exact
test for nominal variables (sex, site, histology, lympha-
denectomy, relapse), and by thev2 test for trend for ordinal
variables (depth of tumor invasion, node metastasis).
Only deaths from gastric cancer were considered as
events in survival analysis, whereas deaths from other
causes were considered as censored observations at thetime of occurrence. Survival curves were computed
according to the Kaplan-Meier method and compared by
the log-rank test. The Cox regression model was used to
evaluate the prognostic significance of positive lavage,
controlling for sex, age, site, histology, depth of tumor
invasion (T), node metastasis (N). The assumptions of
proportional hazard over time made in the Cox model
were met for all the variables tested according to graph-
ical methods.8
A multivariate logistic regression model was used to
evaluate the influence of peritoneal cytology on peritoneal
recurrence, controlling for depth of tumor invasion andnodal metastases. Few explanatory variables were in-
cluded in the logistic models, as the number of events
(peritoneal recurrence) was rather low (n = 33).
RESULTS
In 23 of the 168 cases analyzed (14%), free cancer
cells were detected: 20 positive cases were identified by
immediate cytological examination and 3 additional cases
were detected by immunocytochemistry. Patients with
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positive lavage were more likely to present serosal infil-
tration (100 vs. 46%; P < 0.001) and nodal metastases
(91 vs. 67%; P < 0.001; Table 1).
During the follow-up 21 patients with positive lavage
and 73 patients with negative lavage died from gastric
cancer. Among the latter group, 13 patients died from
other causes. Disease-related survival after 3 years was
50% (95% C.I. 42%59%) in patients with negative lavage
and 9% (95% C.I. 2%27%) in patients with positive la-
vage (P < 0.001; Fig. 1).Mortality risk increased more than threefold in the
presence of peritoneal free cancer cells both in univariate
survival analysis (RR of positive versus negative free
cancer cells = 3.3, 95% CI 2.05.3; P < 0.001) and after
controlling for sex, age, histology, and site of the tumor
(RR = 3.2, 95%CI 1.95.4; P< 0.001). The significance of
this novel prognostic factor could also be appreciated after
controlling for nodal involvement (RR = 2.0, 95% CI 1.2
3.4; P = 0.014), but it disappeared after adjusting for
depth of tumor invasion (RR = 1.2, 95% CI 0.72.0;
P = 0.519; Table 2). Survival analysis was repeated on
patients with serosal invasion (n = 90), who comprised all
cases with positive cytology and most of the disease-re-
lated deaths (75 of 94 patients). In this subgroup perito-
neal lavage lost its statistical significance in the univariate
analysis (RR = 1.5, 95% CI 0.92.5; P = 0.119).
Disease recurrence was observed in 21 patients (91%)
with positive immunocytology and in 74 patients (51%) with
negative lavage(P< 0.001). Relapse during the firstyearof
follow-up was observed in most of the patients with positive
immunocytology (17/23 = 74%) but only in one third of
those with negative lavage (50/145 = 34.5%). In the first
group 16 patients (70%) showed a diffused peritonealrelapse as compared to 19 (13%) in the second group
(P < 0.001). In turn, patients with negative lavage pre-
sented a slightly higher proportion of recurrence at gastric
bed level (n = 27, 19%) and in the liver (n = 19, 13%) with
respect to patients with positive lavage (n = 4, 17% at
gastric bed level and n = 2, 9% at both the hepatic and
peritoneal levels). The sensitivity of peritoneal lavage in
predicting peritoneal recurrence was rather low (16/
35 = 46%), whereas the specificity was much higher (126/
133 = 95%); the positive and negative predictive values
were, respectively, 70% (16/23) and 87% (126/145).
0
10
20
30
40
50
60
70
80
90
100
0 12 24 36 48 60
time (months)
survival(%)
Lavage
Neg. 145 114 77 68 53
P
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As shown in Table 3, peritoneal recurrence was mostly
confined to patients with T3-T4 disease, with positivelavage approximately doubling the risk of recurrence. In
the logistic regression model, peritoneal immunocytology
emerged as the most important predictor of peritoneal
recurrence (Table 4). Peritoneal immunocytology retained
its prognostic value even in the subsample of patients with
serosal invasion (OR of positive versus negative free
cancer cells = 6.4, 95% CI 2.218.8; P< 0.001).
DISCUSSION
The most important findings of the present investigation
are the following:
Positive immunocytology of the peritoneal lavage fluid
was confined to patients with gastric cancer invading
the serosa (pT3/pT4).
Positive peritoneal lavage was the most important
predictor of peritoneal recurrence of thetumor.However,
it is questionable whether this finding represents a real
predictive achievement or a kind of quality control.
Patients with positive peritoneal lavage had a very poor
prognosis. Their cumulative survival dropped to 48%
after 1 year, to 13% after 2 years, and to 9% after 3
years, whereas in patients with negative immunocytol-
ogy, survival was 80%, 55%, and 50%, respectively.
In multivariate survival analysis, peritoneal immunocy-
tology was an independent predictor of mortality when
controlling for sex, age, site, histology, and nodal
metastases, but not when adjusting also for depth of
tumor invasion. Similarly, when considering only the
subgroup of patients with serosal invasion, the influ-
ence of peritoneal cytology on survival was no longer
significant.
The poor prognosis of patients suffering from advanced
gastric adenocarcinoma who undergo curative surgical
operation is still an unsolved problem both in Eastern and
Western countries, because of the high incidence of re-
lapse, especially peritoneal recurrence.1,912 A great
number of studies24,1319 maintain that cytological
examination of peritoneal lavage, when performed
immediately after laparotomy and before every surgical
maneuver, is useful in individuating high-risk patients. In
our survey a positive immunocytology was associated
with a low long-term survival in univariate analysis
Table 2.
Relative risks (with 95% confidence intervals in parentheses) of
death from gastric cancer in the 168 patients who underwent
curative resection (R0). Relative risks and significance of dif-
ferences (Likelihood Ratio Test) were derived from Cox
regression model, controlling for all other variables. Calculation
of the relative risk for age was based on an increase in thevalues of 1 SD
Variables
Relative risk Adjusted
for all other variables P Value
Sex (women versus men) 1.0 (0.71.7) 0.862
Age (SD = 11.8 years) 1.3 (1.01.6) 0.024
Site
Middle third versus
upper third
0.9 (0.61.6) = 0.381
Lower third versus
upper third
0.7 (0.41.2)
Histology
Diffuse versus intestinal 1.0 (0.61.6) 0.906
Nodal involvement
pN1 versus pN0 3.8 (1.68.6)
pN2 versus pN0 4.9 (2.111.5)
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(Fig. 1) and relapsing patients with positive immunocy-
tology showed a progression of illness in most cases, at
the peritoneal level. This examination, even if it is char-
acterized by a high specificity in predicting peritoneal
relapse, had a low sensitivity (46%), in agreement with a
large Japanese study.4
Therefore the technique is oftenused in various centers with some variations aiming at
improving its sensitivity, as immunocytochemistry,20,21 or
together with further analysis for indirect research of
hidden metastasis in the peritoneal lavage fluid, like
carcinoembryonic antigen (CEA) dosage2224 and mRNA
of trypsinogen25 or CEA.26
In the current literature the prevalence of positive
cytology in the peritoneal lavage fluid ranges from 5% to
55%.24,1321 These large fluctuations in positive cyto-
logical diagnoses largely reflect methodological differ-
ences in series recruitment, as some studies considered
only patients with R0 resection whereas others focused
on patients with macroscopic serosal invasion. Some
investigations also included patients with peritoneal
carcinosis and/or ascites, whereas others included early
gastric cancer. Indeed the fluctuations in the percentage
of positive cytology are somewhat blunted (from 12% to
34%) if one takes into account only patients with serosal
invasion.4,14,1618,21 In the present series the prevalence
of positive immunocytology was in the middle of the range
reported in the scientific literature, being 13.7% in the
overall series (n = 168) and 25.5% in patients with
serosal involvement (n = 90). When considering alsopatients with residual tumor or those who died in the
postoperative period, the prevalence of positive immun-
ocytology rose to 17.6% (33/188).
A statistically significant correlation was found between
positive lavage and advanced stage (P< 0.001) and nodal
involvement (P < 0.001), as already reported by some
authors.4,11,15,17,19,21 The most important observation
was that no patients with T1 or T2 tumors had positive
washings, confirming previous studies.3,18,19 However,
one should keep in mind that T1 and T2 subgroups had a
limited size (n = 26 and n = 52, respectively). Positivecytology was always associated with neoplasms infiltrat-
ing the serosa (T3) or adjacent organs (T4), and this
finding seems to attribute once again a crucial role to
infiltration and crossing of gastric serosa as a prerequisite
for the transcelomatic metastasising of gastric carcinoma.
In the present investigation, peritoneal immunocytolo-
gy was a strong predictor of mortality in univariate sur-
vival analysis, in agreement with the current
literature.14,17 However, in multivariate survival analysis
this variable did not emerge as an independent prog-
nostic factor, in contrast with other studies. It should be
pointed out, however, that some of these studies4 in-
cluded among the patients with positive cytology some
patients who also had peritoneal dissemination, whose
prognosis is very poor.
In conclusion, peritoneal lavage is a marker of tumor
progressioni.e., serosal invasionand a strong pre-dictor of mortality in gastric cancer. However, its prog-
nostic significance is already provided by the TNM staging
system in this Italian series. Hence, the insertion of this
novel marker (M1cy+) in the TNM UICC staging, proposed
by some authors,27,28 seems to be premature. Neverthe-
less, as positive immunocytology predicts to a certain
extent peritoneal relapse of the tumor, in spite of its low
sensitivity, this diagnostic technique may be useful in
identifying patients who could benefit from intraperitoneal
treatments, such as peritoneal chemohyperthermia.29,30
ACKNOWLEDGMENTS
This research was supported by a grant from the Italian
Association for Cancer Research (AIRC), regione Veneto.
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