Many factors affect serum tumor markers. Some tumor markers can be elevated in normal patients or non-malignant disease. Stage and tumor burden are the most important factors that affect the tumor markers at diagnosis. Cisplatin and etoposide combination is one of the most favorable choices in the treatment of SCLC. Most studies, which analyzed the association between levels of serum tumor markers and response to therapy in patients with SCLC, were performed in patients who were treated with cisplatin and etoposide combination [8]. Trials of serum tumor markers with other chemotherapeutic drugs were inadequate. We studied tumor markers in SCLC patients who were treated with combination of cyclophosphamide, epirubicin and vincristine.

We researched stage extensively before treatment. We performed stage evaluation with clinical examination, direct radiography, computerized tomography (CT), brain and thoracic scan, unilateral iliac crest bone marrow aspiration, and biopsy, radionuclide bone scan and abdominal ultrasonography and/or computerized tomography. Diagnosis usually was established on biopsy specimen by fiber optic bronchoscope. Liver biopsy or CT guided transthoracal lung biopsy were used if bronchoscopic biopsy was not satisfactory. Serum analysis including complete blood count, electrolyte concentrations, creatine, calcium, alkaline phosphatase, transaminase levels and prothrombin time were performed before treatment. Complete blood count and chemistry analysis was performed before every chemotherapy course.

All patients received cyclophoshamide 750 mg/m² iv day 1, epirubicin 90 mg/m² iv day 1, and vincristine 1 mg/m² (maximum 2 mg) iv day 1 every three week as first line. Twenty-seven patients received Dalteparin 5000 U/day during first line chemotherapy. Dose reduction was performed at the next course as 25% rate of total dose in patients who had grade 4 myelotoxicity. Patients with limited disease underwent local thoracic radiotherapy if they had partial response and stable disease after six-course chemotherapy. Patients with complete remission underwent prophylactic cranial irradiation. All patients who had progression were treated with second line chemotherapy.

We studied the incidence and levels of NSE, LDH, CEA and ferritin in patients’ sera. Tumor markers were determined at diagnosis and after chemotherapy. Marker levels were classified as normal and elevated. NSE and ferritin were analyzed by radioimmunoassay (RIA) (Cis kit for NSE, Icn kit for ferritin), CEA by automated chemiluminescence’s system (Chiron kit), LDH by auto analyzer (Konelap 60) (Dialab kit). Reference ranges were 4.7-14.7 ng/ml for NSE, less than 3.5 ng/ml for CEA, 0-450 U/L for LDH, and 17-390 ng/ml in male, 10-90 ng/ml in female for ferritin.

Statistics
Serum levels of tumor markers at diagnosis and after treatment were compared by Wilcoxon test. The comparison of tumor marker levels at diagnosis between stage groups were performed by Mann Whitney U test. Disease free and overall survival time and survival rate were estimated by using the method of Kaplan Meier and compared by using log rank test.

Table 1: Patients’ characteristics

Table 2: Serum levels of tumor markers at diagnosis according to stage

Fig 1: Rate of patients with elevated serum levels of tumor markers at diagnosis

Table 3: Changes in serum tumor markers after treatment

Fig 2: Distributions of NSE levels according to stage groups

There was no statistically significant difference in CEA, ferritin, and LDH before and after treatment (Table 4). Median level of NSE at diagnosis was 24.4 ng/ml (range 5.0-616.0) and was 13.0 ng/ml (range 4.8-244.0) after chemotherapy in all patients (p=0.0036). Level of NSE decreased significantly in all patients and limited stage subgroups after first line treatment (Table 5, Figure 3). There was no significant difference in extensive stage patients.

Table 4: NSE levels at diagnosis and after treatment according to stage group

Table 5: Response to therapy according to NSE levels*

Fig 3: Changes in NSE levels after treatment with cyclophosphamide, epirubicin, vincristine

We obtained higher response rate for overall objective response without statistically significant difference in patients with normal NSE levels than patients with elevated NSE levels (76.9 versus 52.4, respectively; p=0.58). Median progression-free and overall survival times were higher in patients with normal NSE levels. We calculated median progression-free survival as 11.1±1.47 months (95% CI 8.23-14.01) in patients with normal NSE levels and 8.11±0.96 months (95% CI 6.29-9.99) in patients with elevated NSE levels (p=0.05). Overall survival was median 14.0±1.20 months (95% CI 11.64-16.36) in patients with normal NSE levels and 8.0±0.85 months (95% CI 6.34- 9.66) in patients with elevated NSE levels (p=0.09). Oneyear progression-free and overall survival rates were higher in patients with normal NSE levels but 2-year progressionfree and overall survival rates were not different between two groups (Figures 4 and 5). Survival results were summarized in table 6. No correlation between response to therapy, survival and levels of CEA, Ferritin and LDH were found.

Fig 4: Progression-free survival according to NSE levels by Kaplan Meier test (p=0.05), (— Normal NSE levels, --- Elevated NSE levels)

Fig 5: Overall survival according to NSE levels by Kaplan Meier test (p=0.09), (— Normal NSE levels, --- Elevated NSE levels)

Table 6: Estimated survival time by Kaplan Meier test according to NSE levels

NSE is the most useable tumor marker in lung cancer patients with small cell histology. NSE is the neurol isoenzyme of the intracytoplasmic enzyme enolase, which was first found in the extract of brain tissue and was later shown to be present in neuroendocrine cells and tumors. Elevated serum concentrations of NSE have been found in over 70% of patients with SCLC. In general, studies noted that NSE levels are higher in the patients with extensive stage than limited stage [11-13]. In our study, tumor marker levels were classified as elevated or not elevated. NSE was elevated in 77.6%, CEA in 47.9%, ferritin in 45.8%, LDH in 22.9%. Concentration of NSE in serum was higher with statistically significant difference in extensive disease than limited disease. The serum NSE levels are associated with stage and reflect the tumor burden. After treatment NSE levels decreased significantly in all patients with limited stage disease. NSE levels in patients with extensive stage didn’t decrease significantly. Patients with extensive stage had bad prognosis and combination chemotherapy was less effective than limited stage. Therefore tumor markers could not reflect the response to therapy.

We determined that patients whose NSE levels were not elevated had higher response rate than patients with elevated NSE levels but this difference was not statistically significant. Median progression-free and overall survival times in patients with normal NSE levels were superior to patients with elevated NSE levels. One-year survival rate was also superior in the patients whose NSE levels were not elevated, but 2-year survival rate was similar. Many studies examined the prognostic significance of serum NSE in patients with SCLC [5,14]. Jorgersen at al. [15] showed in 770 patients that NSE was the most powerful predictor of survival followed by performance status and stage of disease. Bonner et al. [8] showed that both pretreatment NSE and treatment induced minimum NSE were independent prognostic predictors of time to progression and survival.

We studied serum tumor markers in patients who were treated with combination of cylophosphamide, epirubicin and vincristine. NSE is correlated with stage and decrement after treatment significantly. NSE levels may play a role as prognostic and predictive factor in patients who were treated with this combination. Although there are many data in the literature about CEA, ferritin, and LDH that are used as biomarkers in patients with SCLC, we did not find any correlation in our patients who were treated with this combination [6,7,16,17]. Comparison of patients who did and did not receive low molecular weight heparin has not revealed further significant results for NSE, CEA, ferritin and LDH. Receiving low molecular weight heparin didn’t affect results of tumor markers analysis. In conclusion; NSE was the most reliable tumor marker in small cell lung cancer patients who were treated with combination of cylophosphamide, epirubicin and vincristine. NSE was in correlation with stage and reflected the response to therapy in patients who were treated with this regimen.

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