The important role of circulating CYFRA21-1 in metastasis diagnosis and prognostic value compared with carcinoembryonic antigen and neuron-specific enolase in lung cancer patients

Background The roles of carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CYFRA21-1) and neuron-specific enolase (NSE) in metastases occurrence and poor diagnosis in specific histological classifications of lung cancer need further exploring. In this study, we investigated relationship between elevated levels of three biomarkers of CEA, CYFRA21-1 and NSE (individually and in combination) and metastasis, survival status and prognosis in lung cancer patients. Methods Eight hundred and sixty eight lung cancer patients including adenocarcinoma (ADC, N = 445), squamous cell carcinoma (SCC, N = 215), small cell lung cancer (SCLC, N = 159) and other types (N = 49) were categorized into negative, moderate and high groups according to serum levels of biomarkers, and were then categorized into negative, single, double and triple groups according to any positive combination of three biomarkers. The cutoff values of three biomarkers for groupings were developed on the training group (N = 432) and verified in a validation group (N = 436). Clinical and laboratory characteristics were then assessed for correlation with occurrence of metastasis, survival status and prognosis between the two groups. Further correlation analyses were also conducted by different subtypes (ADC, SCC and SCLC) and tumor stages (I + II, III and IV) of lung cancers. Results The consistent results between training and validation group confirmed the rationality of grouping methods. CYFRA21-1 levels had stronger association with metastases and survival status than CEA and NSE in all lung cancer patients. When stratified by subtypes, these significances only existed in ADC patients for CYFRA21-1. Cox regression analyses showed that CYFRA21-1 and NSE were independent prognostic factors for lung cancer patients. However, only CYFRA21-1 was an independent prognostic factor in ADC and SCLC patients subtypes. Cox-regression results also indicated that CYFRA21-1 could act as independent prognostic factor in different stages (I + II, III and IV) of lung cancer. Conclusion CYFRA21-1 was more important in metastasis occurrence and in predicting poor prognosis in lung cancer patients than CEA, NSE and positive numbers of biomarkers. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3070-6) contains supplementary material, which is available to authorized users.


Background
Globally, lung cancer has the highest associated mortality among all malignant cancers [1]. The 5-year survival rate in advanced stage cancers is 15%, as compared to 80% in early stage lung cancers [2]. One of the reasons is that most patients are diagnosed at advanced stages due to lack of sensitive and specific early diagnostic biomarkers [3]. Nonsmall-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers; the remaining 15% cases are classified as small cell lung cancer (SCLC) [4]. Although chemotherapy and targeted therapy are the main clinical treatment especially of stage IV patients, yet there is only 4-5% improvement in 5-year survival rates for stage I-III patients, and no significant improvement for stage IV patients [5]. The diagnostic methods include chest x-ray, computed tomography (CT) and needle biopsy of lung [6,7]. However, the high cost and/or invasive nature of these investigations limit the widely use in clinical diagnosis.
During past decades, many advances have been made in the identification of tumor-associated markers in body fluids such as plasma, serum or bio-aerosols such as exhaled breath condensate (EBC) [8,9]which could facilitate early detection and help for treatment monitoring [10]. For lung cancer diagnosis, the leading markers used are carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CYFRA 21-1) and neuron-specific enolase (NSE). CEA, which is closely related to histological classification, is considered valuable for diagnosis of ADC [11]. CYFRA 21-1 and NSE are used for the diagnosis of SCLC [12,13]. Increasing trend in levels of CEA, CYFRA21-1 and NSE have been associated with metastasis and poor prognosis [14][15][16]. However, limitations of previous studies are either in small sample sizes (N = 200-300) or not analyzed in combinations.
In this retrospective study we evaluated the predictive values of serum levels of CEA, CYFRA21-1 and NSE for prognosis and occurrence of metastasis, and the association of these biomarkers with clinical characteristics.

Patients
This study recruited 868 lung cancer patients who were admitted to West China Hospital between 2008 through 2012. All data were obtained from medical records within 2 weeks of diagnosis, and information regarding metastasis was obtained through reports of whole-body CT scan, bone scan, lymph node and fiber optic bronchoscopy biopsy. Survival time was obtained during subsequent follow-up visit or telephonic inquiry. Those patients who did not receive CEA, CYFRA21-1 and NSE determinations and lack of follow-up data were excluded. Data on stage were according to the TNM Classification of Malignant Tumors, 7th Edition [17].
The overall survival time was calculated as time from the date of diagnosis through the date of death or last follow up visit (if the exact date of death was unavailable). Prior to surgery or any other treatments, serum concentrations of CEA, CYFRA21-1 and NSE were measured by immunoassays. Based on the reported literatures, the threshold values for CEA, CYFRA21-1 and NSE levels were 3.4 ng/mL, 3.0 ng/mL and 15.0 ng/mL, respectively [17].

Study design
Depending on the levels of CEA, CYFRA21-1 and NSE, the study subjects were divided into three groups (negative, moderate and high). For CEA analysis, moderate and high groups were defined as 1-10 folds and >10 folds cutoff value, respectively. For CYFRA21-1 analysis 1-3 folds and >3 folds, respectively. For NSE analysis, 1-2 folds and >2 folds, respectively. This analysis was performed in a randomly selected training group (N = 432), reserving the left 436 cases for validation. The cutoff values of three biomarkers for groupings were developed on the training group and tested in a validation group.
Next, we determined the correlations of biomarker levels with three main histological subtypes, ADC, SCC and SCLC. The association analyses of other tumor types (N = 49) such as large cell lung cancers and adenosquamous carcinoma were also performed which showed no positive prognostic value (Data not shown).
Finally, the diagnosis, metastasis and prognostic values of combination patterns of three biomarkers were also evaluated. In brief, patients were grouped as negative, single, double and triple positive of biomarkers. Negative indicated that serum levels of all three biomarkers were below cutoff levels. Single, double, triple positive meant that concentrations of any one, two or all three biomarkers exceeded cutoff values.
Statistical methods SPSS 19.0 for Windows (SPSS Inc, Chicago, USA) was used for data analyses. Chi-square test was performed to evaluate the inter-group differences. Kaplan-Meier test was used to calculate the survival status of different groups, and Logrank test was used to compare the survival among three groups. Multivariate Cox regression analysis was used to determine the clinical characteristics, metastasis and survival status in order to estimate the hazards ratio for different serum levels of CEA, CYFRA21-1 and NSE and identify independent predictors of poor prognosis.
In the present study, 18.3% of all subjects were small cell lung cancer (SCLC) patients. In these patients, we observed a correlation between increased levels of CEA and occurrence of mediastinal and peritoneal metastasis (P < 0.05) (Additional file 4: Table S4A); between increased levels of CYFRA21-1 and liver metastasis (P < 0.05) (Additional file 4: Table S4B); and between increased NSE levels and occurrence of lymph node metastasis (Negative: 42.1%; moderate: 60.1%; high: 77.8%;P < 0.05) (Additional file 4: Table S4C).      Table 4). The application of 3-tier classification to all types of lung cancers revealed that lymph node metastasis was significantly associated with increased levels of biomarkers (ADC P < 0.05; SCC P < 0.001; SCLC P < 0.05) (Additional file 5: Table S5A-C). In ADC and SCC, increased levels correlated with metastasis to both lymph nodes and other organs (Additional file 5: Table S5A-C).

CYFRA21-1 levels correlated with survival in ADC, SCC and SCLC
Kaplan-Meier survival curves were used to analyze mortality at 3-5 years using SPSS19.0. The results of 3-5 year survival analyses indicated that presence of high concentrations of CEA (TA P < 0.01; VA P < 0.01), CYFRA21-1 (TA P < 0.001; VA P < 0.001), NSE (TA P < 0.05; VA P < 0.05) and positive numbers of biomarkers (TA P < 0.001; VA P < 0.01) were closely associated with survival status in both training group and validation groups (Fig. 1a-d).

Multivariate Cox regression analysis to identify poor prognostic factors
We observed a significant correlation between overall survival and CYFRA21-1, NSE and occurrence of metastasis. Compared with negative group, the hazards ratio increased 1.226 in CYFRA21-1 moderate positive group (Confidence Interval [CI]: 0.977-1.537) and 1.647 in CYFRA21-1 high positive group (CI: 1.273-2.130, P < .001) ( Table 5). For NSE, we did not find a significant difference between hazard risk and NSE moderate positive group (HR: 1.010, CI: 0.808-1.263) but the HR increased 1.291 in NSE high positive group compared with that of negative group (CI: 1.032-1.715, P < .05). As expected, occurrence of metastasis was an independent predictor of poor prognosis (HR: 1.291, CI: 1.025-1.625, P < .05) ( Table 5).
The specific histological grade analysis indicated that high and moderate levels of serum CYFRA21-1 significantly correlated with poor prognosis (HR: 1.860, CI:  (Table 6). In SCC and SCLC patients, only occurrence of metastasis was an independent factor for poor prognosis (Table 6).
Lung cancer patients were then divided into three groups according to stages (I + II, III and IV) and Cox regression was conducted to analyze which biomarker could act as independent factor of poor prognosis in specific stage. The results indicated that CYFRA21-1 correlated dramatically with poor prognosis in all stages of lung cancer patients (Stages I-II: HR 3.666 CI: 1.095-12.279, P < 0.05; Stage III: HR 1.919 CI: 1.200-3.071, P < 0.05; Stage IV: HR 1.473 CI: 1.056-2.053, P < 0.05) ( Table 7 A-C), which confirm the importance of CYFRA21-1 in poor prognosis in different stages of lung cancer besides in specific histological classifications.

Discussion
Although several tumor markers for lung cancer have been identified, none of them is specific for lung cancer diagnosis. CYFRA21-1 has been reported as a poor prognostic factor in various cancers, while NSE has been associated with metastasis, and also used for monitoring response to treatment in multiple myeloma. However, these important biomarkers have not been thoroughly investigated in lung cancer patients. In our study, analyses were performed to confirm the correlations between serums CEA, CYFRA 21-1, NSE, as well as the number of positive biomarkers and metastasis and survival status of lung cancer patients.
All patients were randomly divided into training and validation groups to confirm the grouping rationality of this study. In brief, survival curves and associations with clinical characteristics in the validation group were generally similar to those in training group, though there were some non-significant inconsistence in sex and several organs of metastasis. The results indicated that the increased levels of CYFRA21-1 were strongly associated with metastatic sites and histological grades of lung cancers in both training and validation groups. In specific histological subtypes (ADC, SCC and SCLC) analyses, we also found that CYFRA21-1 correlated more closely to metastasis and survival status than CEA and NSE. To our knowledge, these results have not been reported in any of the earlier literatures.
In multivariate Cox regression analysis, only CYFRA21-1 and NSE were found to be independent predictors of prognosis in lung cancer patients. When sub-grouped, only CYFRA21-1was an independent predictor of poor prognosis in ADC (1.86-fold increased risk in high concentration group) and SCLC (1.365-fold increased risk in moderate group) but not CEA and NSE. Finally it was found that CYFRA21-1 could act as independent factor in early (I + II) and advanced stages (III and IV) of lung cancer. Thus, CYFRA21-1 appears to be more important as a prognostic predictor than the other two biomarkers. Several reports have reported the useful roles of CEA in diagnosis of ADC, CYFRA21-1 in SCC and NSE in SCLC [18][19][20][21]. The increased levels of biomarkers are known to correlate with cancer progression, with their sensitivity depending largely on tumor stage and histological classification [22]. In contrast with the previous reports [25], we found no correlation between increased CEA levels and brain metastasis; however, we did observe a correlation with bone, liver, pleural and peritoneal metastases. The inconsistency could be explained by the smaller sample size (approximate N = 300). Research also indicated that high circulating concentrations of CYFRA21-1 and CEA were associated with advanced stages of lung cancer; levels that were two times higher than cutoff value were associated with   stage III and IV lung cancer patients [23]. Although CYFRA21-1 appears to be the most sensitive and specific marker for NSCLC [26], its relationship with different histological lung cancers has largely remained unknown. An earlier report suggested that CYFRA was a more sensitive and specific marker for SCC diagnosis and was found to be of prognostic values in patients with recurrent NSCLC receiving gefitinib therapy [27,28]. In our study, however, high levels of CYFRA21-1 correlated with survival status in ADC and SCLC but not in SCC patients. It also could be used as an independent predictor of poor prognosis in ADC and SCLC patients. Currently, NSE is the most widely used marker for diagnosis and prognosis of SCLC patients [24]. In our study, although positive levels of NSE significantly correlated with survival in SCLC, it did not qualify as an independent predictor for poor prognosis. For those survival data were followed up via outpatient visit, written informed consents were obtained. Part of the survival data were obtained thorough telephone follow-up, the written informed consent could not be available due to the long journey from their resident to our hospital. Under these conditions, only verbal informed consents were obtained from these subjects or their legal guardians.