Ann Thorac Surg 2003;76:260-265
© 2003 The Society of Thoracic Surgeons
Original article: general thoracic
Suppression of the phytohemagglutinin response to lymphocytes is an independent prognosticator in patients with squamous cell carcinoma of the esophagus
Tadahiro Nozoe, MDa*,
Daisuke Korenaga, MDb,
Takefumi Ohga, MDa,
Motonori Futatsugi, MDa,
Yoshihiko Maehara, MDa
a Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
b Department of General Surgery, Fukuoka Dental College, Fukuoka, Japan
Accepted for publication January 17, 2003.
* Address reprint requests to Dr Nozoe, Department of General Surgery, Fukuoka Dental College, 2-15-1, Tamura, Sawara Ward, Fukuoka, 814-0193, Japan
e-mail: nonta{at}college.fdcnet.ac.jp
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Abstract
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BACKGROUND: The preoperative immunological condition of patients with malignant tumors should be considered in determining the prognosis.
METHODS: A lymphoblastic transformation test in which lymphocytes were stimulated with phytohemagglutinin (PHA) was performed for 155 patients with esophageal squamous cell carcinoma (SCC).
RESULTS: Multivariate analysis demonstrated that a lower preoperative PHA response (p = 0.028), as well as the depth of the tumor (p = 0.011), lymph node metastasis (p = 0.0003), lymphatic permeation (p = 0.002), and the incidence of postoperative complication (p = 0.016) were independent factors associated with a poor prognosis for patients with SCC of the esophagus.
CONCLUSIONS: A suppressed PHA response is an additional significant prognosticator for SCC of the esophagus.
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Introduction
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Suppression of the immune system is known to be associated with malignant tumors [1, 2]. Hence, the influence of immunological disorders derived from malignant tumors should be considered along with the direct effects of malignant tumors, especially in cases of highly malignant esophageal carcinoma. Immunological parameters have been shown to have prognostic significance for patients with esophageal carcinoma [3–6].
The phytohemagglutinin (PHA) response test, which measures immune system dynamics serologically, is performed to examine potential of lymphoblastic transformation under the principle that lymphocytes incubated with a nonspecific mitogen such as PHA are known to acquire the capacity to differentiate and transform into lymphoblasts. The clinical application of PHA response as a prognostic marker has been reported in patients with carcinomas of the stomach [7], colorectum [8], and breast [9].
In the current study, we sought to clarify the significance of PHA response of leukocyte blastogenesis as an indicator of prognosis of the patients with squamous cell carcinoma of the esophagus.
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Patients and methods
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Patients
We consecutively enrolled 155 patients with esophageal squamous cell carcinoma (SCC) who had been treated by esophageal resection and reconstruction of the digestive tract in our institute between 1990 and 1998. These 136 men and 19 women had a median age of 62 years (range, 36 to 89 years).
The patients with advanced esophageal tumors have usually been treated with preoperative induction therapies. Advanced tumors without obstruction due to the tumors, which would prevent the insertion of the applicator for hyperthermic therapy, have been treated with hyperthemia combined with chemotherapy and radiation (HCR). Preoperative induction therapies were provided to 112 patients according to our original protocol [10]. The regimens for the hyperthermia, chemotherapy, and radiation therapies are shown in Figure 1.
Hyperthermia was applied using a frequency system with an endotract electrode (Endoradiotherm 100A, 200A [Olympus, Tokyo, Japan]). A long electrode was inserted into the lumen of the esophagus and placed on the esophageal tumor, and a wide counter electrode was installed on the body surface. Temperature imposed on the tumor was indicated by a thermosensor attached to the balloon surface of the electrode. Hyperthermia was induced in the tumors at 42.5°C to 44.0°C for 30 minutes per treatment for six courses of treatment. Patients who underwent chemotherapy received either 5-mg doses of bleomycin administered six times (total of 30 mg bleomycin) or 40 mg/m2 of cisplatin administered three times (total of 120 mg/m2). Patients who underwent irradiation received 2-Gy doses 5 days per week for 3 consecutive weeks (30 Gy total). Patients who underwent both hyperthermia and chemotherapy received them simultaneously. When hyperthermia and chemotherapy were combined, patients who underwent hyperthermia, chemotherapy, or both after irradiation received the treatment 1 hour after irradiation.
The follow-up for patients was continued until their death, and only patients who died of esophageal SCC were included in the tumor-related deaths. The interval of the follow-up after operation ranged from 29 days to 8 years and 9 months, with a mean of 3 years.
Serum (PHA) response
All blood samples were collected just before the surgical treatment and were immediately processed to measure PHA response. Lymphocyte blastogenesis was stimulated by exposure to phytohemagglutinin (PHA-P [Difco, Detroit, MI]) for 72 hours, and peripheral mononuclear cells were incubated in culture media with and without 0.125% PHA-P for a further 72 hours. The ratio of DNA recoveries, representing PHA response, was expressed as a percentage calculated by the formula: DNA from cells cultured with PHA/DNA from cells cultured without PHA x 100. Values less than 180%, the median value of all results, were considered to be lower PHA responses.
Pathological features were presented according to the guidelines for clinical and pathologic studies on carcinoma of the esophagus established by the Japanese Society for Esophageal Diseases [11], and clinical stages were determined by the TNM classification of malignant tumors prescribed by the International Union Against Cancer [12]. Clinicopathological features were compared between patients with lower PHA responses and those with higher PHA response.
Statistical analysis
The 
2 test and Student’s t test were used to compare the clinicopathological data. The cumulative survival rates were calculated by the Kaplan-Meier method, and the survival curves were tested by the Mantel-Cox method. The multivariate survival analysis was calculated according to Cox’s proportional hazards model in a forward stepwise manner. A p value of less than 0.05 was considered significant.
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Results
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A lower PHA response level was observed in 79 of the 155 patients (51.0%, group L). A higher PHA response level was observed in the remaining 76 patients (49.0%, group H).
The preoperative therapies and surgical treatments are shown in Table 1.
The proportion of patients who had been treated with preoperative therapies was significantly higher in group L (81.0%, 64 of 79) than in group H (55.3%, 42 of 76; p = 0.0006). There was no significant difference between the groups regarding surgical procedures.
The clinicopathological features of the patients are shown in Table 2.
No significant differences between the groups were observed regarding gender and age of the patients, location, degree of differentiation, and mean size of the tumors. Although the depth of the tumors was significantly more progressive in group L than in group H (p = 0.029), there was no significant difference between the two groups regarding the proportions of lymph node metastasis, lymphatic invasion and venous invasion, and TNM stage of the tumors. No significant differences were observed between the two groups regarding curability and the incidence of postoperative complications such as anastomotic breakdown and pulmonary diseases. One-, 3-, and 5-year survival rates of patients with lower PHA response were 77.4%, 46.0%, and 41.6%, respectively, which were significantly worse than those of patients with higher PHA response (87.4%, 71.0%, and 63.8%, respectively; p = 0.005) (Fig 2).
According to multivariate analysis, depth of the tumor (p = 0.011), lymph node metastasis (p = 0.0003), lymphatic permeation (p = 0.002), incidence of postoperative complication (p = 0.016), and lower PHA response (p = 0.028) were found to be factors independently associated with worse prognosis of the patients (Table 3).
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Fig 2. Survival curves. The prognosis of patients with the lower phytohemagglutinin (PHA) response (group L, thick line) was significantly more unfavorable than that of patients with the higher PHA response (group H, thin line; p = 0.005).
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Additional analysis was performed restricted to 106 patients who had been treated with additional preoperative therapies. No significant differences were found regarding surgical treatments (Table 4)
or clinicopathological features of the patients (Table 5). Likewise, 1-, 3-, and 5-year survival rates of patients with lower PHA response were 73.6%, 38.3%, and 33.2%, respectively, and they were significantly worse than those of patients with higher PHA response (84.3%, and 67.6%, and 59.9%, respectively; p = 0.010) (Fig 3).
Multivariate analysis demonstrated that lower PHA response (p = 0.011) as well as lymph node metastasis (p = 0.002) and lymphatic permeation (p = 0.015) were factors independently associated with worse prognosis of the patients who had been treated with preoperative therapies (Table 6).
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Fig 3. Survival curves of patients who had been treated with preoperative therapies. The prognosis of patients with the lower phytohemagglutinin (PHA) response (group L, thick line) was significantly more unfavorable than that of patients with the higher PHA response (group H, thin line; p = 0.010).
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Comment
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Although recent progress in perioperative management, including assessment of preoperative conditions, has undoubtedly contributed to the remarkable improvement of the prognosis of patients with esophageal carcinoma [4, 13, 14], the prognosis still remains dismal. Furthermore, it has been noticed that preoperative impaired immunity associated with esophageal carcinoma might account for its notoriously malignant potential and unfavorable prognosis of the patients [3–6].
We have emphasized the clinical impact of serum C–reactive protein (CRP) levels in patients with malignant tumors, and have reported the significance of preoperative serum CRP elevation as an indicator of an unfavorable prognosis in carcinomas of the esophagus [3] and colorectum [15]. Preoperative elevation of serum CRP was also closely correlated with impaired immunity associated with decreased percentage of lymphocytes in the peripheral blood in patients with colorectal carcinoma [16]. Furthermore, patients with esophageal SCC who had a lower prognostic nutritional index value, which was established to evaluate the immunological and nutritional condition of patients with gastrointestinal diseases [17, 18], were found to have a significantly more unfavorable prognosis [4]. These investigations have suggested that a preoperative immunological assessment might also provide useful information regarding the prognosis of the patients with esophageal carcinoma.
The lymphoblastic transformation test, in which lymphocytes in the peripheral blood are stimulated with PHA, is a simple, inexpensive method for examining the immunological condition of patients. The clinical significance of a preoperative evaluation of the PHA response in patients with esophageal carcinoma has not been fully investigated. Bentdal and colleagues [19] reported that lymphocyte responses to PHA were significantly lower in patients with inoperable esophageal carcinoma, which suggests a close correlation of PHA response with the invasive extent of esophageal carcinoma. Saito and colleagues [20] reported that the PHA response measured immediately before surgery was significantly lower in patients with esophageal carcinoma who had fatal septic complications after surgery, which might indicate the clinical usefulness of PHA response for predicting the incidence of postoperative morbidity. Tsutsui and colleagues [5] previously found that low values in all of three immunological parameters, natural killer cell activity, CD4+/CD8+ ratio, and PHA response, were independent indicators of poor prognosis of patients with esophageal carcinoma. In the current study, a lower preoperative PHA response was found to be an independent prognosticator for patients with SCC of the esophagus.
Patients with esophageal carcinoma who had been treated with preoperative therapies such as irradiation, chemotherapy, hyperthermia, or combinations of these modalities may have greater immunity impairment. In the current study, patients who had received preoperative treatments had a significantly greater proportion of lower PHA value. This result is consistent with that in a previous investigation reported by Tsutsui and colleagues [6]. Moreover Heidecke and colleagues [21] have recently reported that chemoradiotherapy performed for patients with esophageal SCC caused subsequent suppression of T lymphocyte functions. Thus, a decrease in the PHA response might result from the preoperative induction therapies as well as from tumor aggression. Therefore, if so, preoperative induction therapy would be actually available only when the benefit of the antitumor effect would exceed the detriment of the resulting impaired immunity derived by the preoperative therapies. Although, in the current study, the existence of preoperative induction therapy was found not to be an independent prognostic indicator of esophageal SCC. We demonstrated that suppression of the PHA response to lymphocytes was a significant independent prognostic indicator of esophageal SCC.
We have previously reported that patients with esophageal SCC who received ineffective preoperative therapy had a significantly poorer prognosis than those who did not receive any preoperative therapy [22]. Hence, an appropriate determination whether preoperative therapy is indicated as well as an assessment for preoperative immunological aspects of patients are essential for improving the prognosis in esophageal SCC.
Although no significant correlation was observed between a lower PHA response and the incidence of postoperative complications, both lower PHA response and the incidence of postoperative complications other than those involving tumor-related factors such as tumor depth, lymph node metastasis, and lymphatic invasion were found to be independent prognostic indicators for patients with esophageal carcinoma. This finding was consistent with the results presented in previous reports [4, 23].
The results of the current study showed that suppressed PHA response, an immunological parameter, was a significant independent indicator of an unfavorable prognosis for patients with SCC of the esophagus. Clinical efforts to improve the immunological condition are required to acquire a more favorable prognosis for these patients.
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References
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Abstract
Introduction
