The Prognostic and Predictive Value of Pretreatment Serum Tumor Markers in Lung Adenocarcinoma with Different EGFR Status

Background: The prognostic value of carcinoembryonic antigen (CEA), cytokeratin-19 fragments (Cyfra21-1), neuron-specic enolase (NSE), Glycogen Antigen 153, Glycogen Antigen 199 has been investigated in Lung Adenocarcinoma (LUAD). However, few studies have directly focused on the association between serum tumor markers and epidermal growth factor receptor (EGFR) mutation status. Material and Method: 146 patients with stage IIIB and IV LUAD were enrolled between 2008 and 2018. Correlations between serum tumor marker levels, EGFR mutations and survival were analyzed and prognostic factors were identied. Result: Of eligible 90 patients with EGFR-mutant LUAD, CA 15-3 (7 versus 9 months, 0.037 for PFS; 25 months versus 36 month, P = 0.003 for OS, ) and Cyfra21-1 (7.0 versus 9.0 months, P =0.010 for PFS, 25 months versus 36 months, P=0.044 for OS,showed negatively signicant correlation with overall survival and Progression free survival. For parents without EGFR-mutation, CA125 (month versus 8 months, P = 0.013 for DFS, 18 months versus 24 month, P = 0.016 for OS) also showed negatively signicant correlation with overall survival and Progression free survival. Conclusion: CA153 and Cyfra21-1 was a prognostic serum biomarker in EGFR-mutant LUAD patient, coincide with CA125 in EGFR-WT LUAD group.Cyfra21-1 levels was also a predictive serum biomarker for in EGFR-mutant LUAD patient who received EGFR-TKI treatment.


Background
The lung cancer in the morbidity and mortality was ranking rst in various cancers worldwide. Only 10-15% of patients survive 5 years from diagnosis. Advanced NSCLC is fatal in 100% of cases (1), Lung adenocarcinoma (LUAD), which accounts for approximately 40% lung cancer, could be divided into EGFRmutated LUAD group and EGFR-wild type LUAD group (2). EGFR-TKI therapy is a standard of treatment in advanced EGFR-mutant LUAD patients. EGFR exon 19 deletion (del19) and exon 21 Leu858Arg substitution (L858R) make up around 90% of all EGFR mutation-positive lung adenocarcinomas, which is closely associated with disease control rate and progression free disease (PFS) to EGFR tyrosine kinase inhibitors (3)(4) Measurement of serum tumor markers is a non-invasive means to monitoring e cacy of treatment and assess prognosis in cancers (5). Actually, serum cytokeratin 19 fragments (CYFRA 21 − 1), carcinoembryonic antigen (CEA) or neuron-speci c enolase (NSE), Carbohydrate antigen 19 − 9, Carbohydrate antigen 125, Carbohydrate antigen 15 − 3 were used to be an independent prognostic marker and in non-small cell lung cancer (6-8). Some studies showed high level of serum Cytokeratin 19 was negatively correlated with Overall survival in LUAD (9), Other studies showed that Cytokeratin 19 level in serum has no prognostic value in patients with adenocarcinoma (10) CEA-high level was easier observed in LUAD harbored EGFR-mutation, but not be correlated with the EGFR-TKI effects of treatment (11). On the Contrary, pretreatment serum Tumor biomarker level such as C CYFRA 21 − 1 was a predictive marker of EGFR-TKI treatment in EGFR-mutated NSCLC patients (12). These data indicate that those tumor biomarkers may function differently in the initiation and progression of EGFR-mutant LUAD and EGFR-negative LUAD.
Herein, we identi ed that CYFRA 21 − 1 and CA153 was crucial in the prognostic of unrespectable LUAD patients with EGFR-mutated, while CA125 was of importance in the prognostic of those with EGFRwildtype.

Material And Methods
Patient's enrollment Between April 2011 and April 2018, were diagnosed in Daping hospital and the EGFR status of these people was determined by DNA sequencing or qPCR. Diagnosis of LUAD was staged according to the TNM classi cation of the Union for Inter`national Cancer Control (8th edition).This study was approved by Daping Ethics Committee and conducted abided by the principle of the Declaration of Helsinky; a written consent was informed all patients. Primary treatment of was chemotherapy, radiotherapy, molecular targeting treatment either alone or in combination. Patients with EGFR-mutated received EGFR-TKI drugs were evaluated every 2 months by chest CT scans before treatment and then after 4 cycles of treatment. The tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST verus 1.1) (12). Progression disease free and overall survival was collected directly from the outpatient clinic records or from family contact. All the patients were followed at least two years.

Statistical analysis
Average value between groups was compared by using rank sum test for analysis. Progression-free survival (PFS) is de ned as the time from the date of starting EGFR-TKI treatment to the date of tumor progression, the date of death or nal follow-up. Overall survival (OS) was measured from the beginning of the date of diagnosis to death or the last follow-up. PFS and OS were determined the Log-rank test and by the Kaplan-Meier method for univariate analysis. And The Cox proportional hazards regression model was used to estimate the 95% con dence intervals (CIs) and hazards ratios (HR) in the univariate and Page 4/23 multivariate survival analyses. Data were analyzed using SPSS23.0 software (IBM Corp., Armonk, NY, USA). P values < 0.05 were considered statistically signi cant.

Patient Characteristics
We retrospectively analyzed 146 patients diagnosed stage IIIB-C or IV lung adenocarcinoma at Daping tumor Center General Hospital between April 2011 and April 2018. The clinic pathological characteristics were shown in Table 1. The population were included 73 males and 73 females, with a meanage of 51.1 years (range from 28 years to 80 years). Of 146 patients, 21 (14.38 %) patients had stage IIIB-C disease, and EGFR Mutations were detected in 61.64% (90/146) adenocarcinoma cases. Among the 90 EGFR mutations, 37 were 19 exon del and 40 were L858R. Of the whole study population, 75 (51.37%) were bone metastasis and 46(31.51%)were brain metastasis. At the end of the last follow up, 100 patients had died and the median overall survival was 26.9 months, and the 2-year survival rate was 48.63 % for the whole group   Table 2). Furthermore, we found the patients with high level of NSE showed shorter PFS as compared with low level of NSE ( Supplementary Fig. 1).
Correlation ship of increased CA125/CYFRA21-1/CA153 with PFS and OS based on EGFR mutation status.
In order to explore serum tumor biomarker in LUAD with different EGFR status, the population were divided into two groups: EGFR-mutant LUAD and EGFR-wild type LUAD We found that the positive rate of CEA was signi cantly higher in EGFR-MT LUAD than that in EGFR-WT LUAD (63.33% versus 46.55%,P = 0.027). However No signi cantly difference in others were observed in EGFR-MT LUAD group as compared with EGFR-WT NSCLC group (Table 4).  Figure   2A; 24 months versus 39 month, P = 0.006 for OS, Fig. 2A; CYFRA21-1: 6.0 versus 15.0 months, P = 0.008 for PFS, Fig. 2C; 31 months versus 43 months, P= 0.017 for OS, Fig. 2B; Table 4). Abnormal CA125 levels were not correlated with DFS or OS in EGFR-mutated NSCLC ( Fig. 2C; Table 4).

Discussion
Advanced LUAD is a heterogeneous and complex disease with poor prognosis. However, due to the discovery of EGFR tyrosine kinase inhibitors, EGFR mutation subgroup shows longer progression free disease rate and over survival rate than EGFR-WT subgroup (14).Furthermore, serum tumor markers have been proven associated with EGFR mutation status and e cacy of EGFR-TKI treatment in lung cancer (15) However, there is no a consensus regarding appropriate tumor markers to distinguish the prognosis and e cacy of EGFR-TKI in advanced LUAD patients with different EGFR status. Herein, we screened and identi ed that CA153 and Cyfra21-1 was a prognostic marker in EGFR-mutant LUAD patient,coincide with CA125 in EGFR-WT LUAD group.
CA15-3, a tumor antigen recognized by two monoclonal antibodies DF3 and 115D8, was rstly a biomarker for breast cancer since 1980s (16). In previous study, the sensitivity and speci cities of CA15-3 was lower than other serum biomarkers, such as CEA and CYFRA21 in LUAD (17). Furthermore, CA153 in pleural uid was signi cantly highly expressed than that in serum in the lung cancer 18 , indicating CA153 was of importance in distinguish the cancer patient. However,some other studies shows CA153 does not improve the diagnostic e cacy in lung cancer 19 , indicating CA153 play a complex role in lung cancer. Herein, we rstly reported that high level of CA153 in serum shows longer than that with low in EGFR-mutant LUAD, while not in EGFR-WT LUAD. Meanwhile, Cyfra21-1 was a prognostic serum marker in EGFR-mutant LUAD patient (20), the reduction in serum level of CYFRA21-1 was a reliable biomarker to predict immunotherapy e cacy in NSCLC patients, In our study, we found the pretreatment of Cyfra21-1 was a predictive serum biomarker for EGFR-TKI treatment.
EGFR mutations occurring in the kinase domain are strongly associated with EGFR-TKI sensitivity.
However, subsequent studies revealed that this relationship was not perfect and various predictive markers such as KRAS mutation, EGFR gene copy number have been reported (21). Given the level of tumor biomarkers was correlated with survival time, we highly suspected that the tumor biomarkers could be associated with EGFR-TKI sensitivity (22). Our research concluded that among the above six tumor biomarkers, only the level of CYFRA21-1 can show whether patients with EGFR-mutant are sensitive to EGFR-TKI drug, suggesting that Chemotherapy, apart from EGFR-TKI treatment, might be added to the rst treatment of patients with high level of CYFRA21-1 and advanced EGFR-mutation LUAD (23) CA125, a heavily glycosylated protein and was rst identi ed as an ovarian cancer antigen, was increased in NSCLC but decreased in rectal cancer (24) It was reported that preoperative serum CA125 levels were related with TNM stage in operable NSCLC and was to monitor the tumor recurrence and disseminated failure post operation (25) However, other research suggested that elevated serum CA125 was not correlate with tumor recurrence(26), indicating CA125 plays a complex role in different gene background of lung cancer. To our knowledge, we described that CA125 was closely related with poor prognosis in EGFR-Wild LUAD, not in EGFR-mutant LUAD.
In conclusion, CYFRA21-1 and CA153 were two prognostic markers in unrespectable adenocarcinoma patients harboring EGFR mutations; However, CA125 was an independent prognostic factor only for EGFR wild-type adenocarcinoma patients. Furthermore, the level of CYFRA21-1 was also a predictive maker for EGFR-TKI treatment, of course, a prospective clinical trial is necessary to testify our present ndings.

Conclusions
In common tumor biomarker,we screened that CYFRA 21 − 1 and CA153 was crucial in the prognostic of unrespectable LUAD patients with EGFR-mutated, while CA125 was of importance in the prognostic of those with EGFR-wildtype.

Declarations
Ethics approval and consent to participate This study was approved by the Ethical Committee of Daping Hospital of the Third Military Medical University. We got the agreement and signed consent form of the patient reported in our paper.

Consent for publication
The patients included in this study allowed this paper to include some information of their disease for publication.

Availability of data and materials
All data generated or analysed during this study are included in this published article [and its supplementary information les].

Competing interests
The authors declare that they have no competing interests.