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. 2020 Jun 5;15(6):e0233687. doi: 10.1371/journal.pone.0233687

Prognostic value of postoperative serum carcinoembryonic antigen levels in colorectal cancer patients who smoke

Chih-Sheng Huang 1,2,*, Chin-Yau Chen 1,2, Li-Kuo Huang 2,3, Wei-Shu Wang 2,4, Shung-Haur Yang 1,2,5
Editor: Jason Chia-Hsun Hsieh6
PMCID: PMC7274431  PMID: 32502149

Abstract

Serum carcinoembryonic antigen (CEA) levels can help predict the prognosis of colorectal cancer patients. Accordingly, high preoperative CEA levels that is not restored after surgery are indicative of a worse outcome. On the other hand, smoking can increase serum CEA levels independently of the disease status. Thus, we aimed to evaluate the impact of smoking on the prognostic value of serum CEA levels. This retrospective cohort study included 273 patients who underwent curative resection for stage I–III colorectal adenocarcinoma at a single institution, between January 2010 and December 2017. Patients were grouped as follows: group A, normal preoperative and postoperative CEA levels (n = 152); group B, elevated preoperative CEA levels that returned to reference values after surgery (n = 69); and group C, elevated postoperative serum CEA levels (n = 52). Patients were also grouped according to their smoking history: group S (current smokers, n = 79) and group NS (never and former smokers, n = 194). Group A showed a higher 3-year disease-free survival (DFS) rate (84.9%) than groups B (75.4%) and C (62.0%) (p < 0.001). Postoperative serum CEA levels were significantly higher in the S group than in the NS group (2.6 vs. 3.1 ng/mL, p = 0.009), whereas preoperative levels were similar (3.8 vs. 4.1, p = 0.182). Further, smokers showed higher 3 year-DFS rates than nonsmokers in group C (83.3% vs. 43.9%, p = 0.029). This suggests that while elevated postoperative CEA levels are associated with lower DFS rates in never and former smokers, they are not associated with lower DFS rates in current smokers. We conclude that persistent smoking alters the prognostic value of postoperative serum CEA levels in colorectal cancer patients and that, consequently, alternative surveillance strategies need to be developed for colon cancer patients with smoking habits.

Introduction

Serum carcinoembryonic antigen (CEA) is the most widely used tumor marker for patients with colorectal cancer. Most published guidelines, including those from the National Comprehensive Cancer Network and the American Society of Oncologists, recommend postoperative serum CEA testing every 3–6 months [1,2]. Preoperative CEA levels ≥5.0 ng/mL have been reported to adversely impact survival, independently of tumor stage [3–5]. In 2000, the Colorectal Working Group of the American Joint Committee on Cancer (AJCC) recommended modifications of the TNM staging system to differentiate between tumors of patients with normal vs. elevated serum CEA levels at presentation [6]. However, serum CEA levels are no longer included as a factor for staging in the AJCC TNM staging system 8th edition.

High preoperative serum CEA levels do not return to reference values after surgery in approximately one-third of the patients with colorectal cancer. This indicates the presence of persistent disease and the need for further evaluation [7,8]. Therefore, consideration of both preoperative and postoperative serum CEA levels might effectively predict the prognosis of patients with colorectal cancer [9].

Although CEA is a recognized tumor marker, its levels can be influenced by many factors [10–12]. Tobacco use is one of the most common causes of CEA elevation [13–15], which can lead to inaccurate cancer diagnosis and prognosis. This study aimed to determine the effect of cigarette smoking on the prognostic value of serum CEA levels. We hypothesized that current smokers with elevated postoperative CEA levels might not have lower disease-free survival (DFS) rates.

Materials and methods

After obtaining the institutional review board of National Yan-Ming University Hospital’s approval (NYMUH IRB No.2020A001) and a waiver of the requirement for patient consent, prospectively maintained databases were queried for all consecutive studies.

Patients

A total of 444 patients with colorectal adenocarcinomas received curative treatment at the National Yang Ming University Hospital between January 1, 2010 and December 31, 2017. We excluded 171 patients from the study because of stage IV disease (n = 52), loss to follow-up (n = 26), diagnosis of carcinoma in situ (n = 29), and incomplete CEA data (n = 64). Thus, our study ultimately comprised 273 patients.

Patients were grouped according to their CEA status as follows: group A, normal (<5.0 ng/mL) pre- and postoperative serum CEA levels (n = 152); group B, elevated (≥5.0 ng/mL) preoperative but normal postoperative serum CEA levels (n = 69); and group C, elevated postoperative serum CEA levels (n = 52). The patients were also grouped according to their smoking history. Group S comprised current smokers, defined as people who have smoked in their lifetime and currently smoke cigarettes (n = 74). Group NS comprised never smokers (defined as people who have never smoked) and former smokers (defined as people who have smoked in their lifetime but had quit smoking before surgery) (n = 199, 173 never smokers and 26 former smokers).

Data collection

We prospectively developed a computerized database at our hospital and updated it constantly. The recorded variables included the patients’ demographic data and major comorbidities; family history of cancer; tumor location, number, and stage; macro- and microscopic pathological characteristics; and patient status at the last follow-up.

Evaluation and treatment

Tumor staging was based on the TNM system described in the 7th edition of the International Union Against Cancer/AJCC [16]. Serum CEA levels were measured in a single laboratory using an Elecsys E170 analyzer (Roche Diagnostics, Indianapolis, IN, USA), with a recommended upper reference limit of 5 ng/mL. Preoperative serum CEA levels were measured immediately before the surgery, and postoperative serum CEA levels were measured 4 to 6 weeks after surgery. All patients were evaluated via staging workups including a colonoscopy, complete blood count, serum CEA determination, chest radiography, and computed tomography (CT) of the abdomen.

All patients underwent radical surgical resection. Twenty-two patients also underwent preoperative neoadjuvant chemoradiation therapy (CRT) for locally advanced rectal cancer; preoperative CEA was determined before initiating CRT. The CR protocol was described in our previous study [17]. Postoperative adjuvant chemotherapy was considered for 104 patients with pathologic stage III disease. Of these, 12 did not receive adjuvant chemotherapy due to refusal or poor performance status. The chemotherapy regimens were 5-fluoruracil/leucovorin/oxaliplatin (FOLFOX) in 69 patients, capecitabine/oxaliplatin (XELOX) in 2 patients, oral tegafur/uracil (UFUR) in 19 patients, and oral capecitabine in 2 patients. Postoperative adjuvant chemotherapy was also administered to 80 patients with both pathologic stage II disease and other risk factors such as pathologic stage pT4, lymphovascular invasion, perineural invasion, and anastomosis leakage. The regimen was FOLFOX in 37 patients and oral UFUR in 43 patients.

Surveillance protocol

All patients were followed-up in the outpatient department every 3 months in the first 2 years, every 6 months in the third and fourth year, and annually thereafter. The follow-up examinations included determination of serum CEA levels, chest and abdominopelvic CT, and colonoscopy. It is our policy to perform the first follow-up colonoscopy 6 months after surgery in patients in whom a complete colonoscopy study had not been or could not be performed before surgery. If the patient had undergone complete colonoscopy before surgery, the first follow-up colonoscopy was performed 1 year after surgery.

Statistical analysis

Optimal cutoff CEA values were determined using receiver operating characteristic (ROC) curve analysis and Youden’s index. Chi-square test, Mann-Whitney U test, and Kruskal-Wallis analyses were used to analyze categorical and continuous variables, respectively. Survival curves were plotted using the Kaplan-Meier method, and survival values were compared using the log-rank test. Death and disease recurrence were treated as events in the analysis. Differences in DFS rates in the univariate analysis were assessed using the log-rank test. Hazard ratios and associations with DFS were determined via multivariable Cox regression analysis. Variables with p <0.05 on univariate analysis were included in the multivariable model. Data were analyzed using MedCalc statistical software version 19.0.3 (MedCalc Software bvba, Ostend, Belgium), and p <0.05 was considered statistically significant.

Results

Patient characteristics

Overall population

Out of the 273 patients included in our study, 149 (54.6%) were men. The median age was 71 years (range 28–93 years), and the median pre- and postoperative serum CEA concentrations were 3.9 ng/mL (range, 0.6–263.5 ng/mL) and 2.7 ng/mL (range 0.5–84.9 ng/mL), respectively. Tumors were located in the right colon in 99 patients (36.3%), in the left colon in 101 patients (37.0%), and in the rectum in 73 patients (26.7%).

The median follow-up interval was 46 months (range, 4–117 months). Tumors recurred in 58 patients (21.2%) before the last follow-up. The sites of tumor recurrence were the liver (n = 24), lungs (n = 22), peritoneum (n = 9), para-aortic lymph nodes (n = 9), bone (n = 4), and brain (n = 2). Local recurrence was observed in 13 patients. The 3-year and 5-year DFS rate for all patients was 78.0% and 69.1%, respectively.

By CEA status

The demographic and clinical features of patients grouped according to their CEA status (group A: normal pre- and postoperative serum CEA levels, group B: elevated preoperative and normal postoperative serum CEA levels, and group C: elevated pre- and postoperative levels) are shown in Table 1. There were no significant differences in sex distribution, median age, tumor location, histologic differentiation, or lymphovascular and perineural invasion status among the three groups. Group B and group C patients tended to have a more advanced T and N stage than did group A patients. The percentage of current smokers was higher in group C (46%) than in group A (24%) and B (20%) (p = 0.002).

Table 1. Clinical characteristics by CEA status.
Group A (n = 152) Group B (n = 69) Group C (n = 52) p
Sex
Male 80 (53%) 36 (52%) 33 (53%) 0.239
Female 72 (47%) 33 (48%) 19 (37%)
Age (years), median (range) 70 (32–93) 69 (28–89) 72 (53–88) 0.493
Tumor location
Right colon 52 (34%) 22 (32%) 25 (48%) 0.222
Left colon 59 (39%) 24 (35%) 18 (35%)
Rectum 41 (27%) 23 (33%) 9 (17%)
Preoperative CEA levels (ng/mL), median (range) 2.6 (0.6–4.9) 8.6 (5.0–97.1) 8.9 (2.8–263.5) < 0.001
Postoperative CEA levels (ng/mL), median (range) 2.2 (0.5–4.9) 2.6 (0.8–4.7) 6.4 (5.1–84.9) < 0.001
T Stage
T1 22 (14%) 2 (3%) 2 (3%) < 0.001
T2 32 (21%) 4 (6%) 5 (10%)
T3 94 (62%) 60 (87%) 39 (75%)
T4 4 (3%) 3 (4%) 6 (12%)
N Stage
N0 109 (72%) 34 (49%) 26 (50%) 0.002
N1 33 (22%) 21 (31%) 19 (37%)
N2 10 (6%) 14 (20%) 7 (13%)
TNM stage
I 47 (31%) 5 (7%) 7 (13%) < 0.001
II 62 (41%) 29 (42%) 19 (37%)
III 43 (28%) 35 (51%) 26 (50%)
Differentiation
Well 6 (4%) 0 4 (8%) 0.210
Moderately 142 (93%) 68 (99%) 46 (88%)
Poorly 4 (3%) 1 (1%) 2 (4%)
LVI
Yes 90 (59%) 42 (61%) 31 (60%) 0.973
No 62 (41%) 27 (39%) 21 (40%)
PNI
Yes 18 (12%) 11 (16%) 6 (12%) 0.667
No 134 (88%) 58 (84%) 46 (88%)
Chemotherapy regimen
FOLFOX 54 (36%) 36 (52%) 16 (31%) < 0.001
XELOX 0 (0%) 2 (3%) 0 (0%)
UFUR 35 (23%) 19 (28%) 18 (34%)
Capecitabine 0 (0%) 0 (0%) 2 (4%)
No 63 (41%) 12 (17%) 16 (31%)
Smoking
Yes 36 (24%) 14 (20%) 24 (46%) 0.002
No 116 (76%) 55 (80%) 28 (54%)
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Data are presented as n (%), unless otherwise indicated.

PNI, perineural invasion; LVI, lymphovascular invasion; TNM, tumor-node-metastasis

By smoking history status

The demographic and clinical features of patients grouped according to their smoking history status [smoking (S) and nonsmoking (NS) patients] are shown in Table 2. Compared with the NS group, the S group contained significantly more men (42% vs. 88%, p < 0.001) and younger patients (median age, 73 vs. 68 years, p = 0.009). Postoperative serum CEA levels were significantly higher in the S than in the NS group (2.6 vs. 3.1 ng/mL, p = 0.009), whereas preoperative serum CEA levels were similar (3.8 vs. 4.1 ng/mL, p = 0.182) between groups. More patients had elevated postoperative serum CEA levels in the S than in the NS group (32.4% vs. 14.1%, p < 0.001). There were no significant differences in the pathological characteristics or the 3-year DFS rate between groups (80.2% vs. 77.1%, p = 0.485).

Table 2. Patient characteristics by smoking status.
NS (n = 199) S (n = 74) p
Sex
Male 84 (42%) 65 (88%) < 0.001
Female 115 (58%) 9 (12%)
Age (year), median (range) 73 (32–93) 68 (28–87) 0.009
Tumor location
Right colon 81 (41%) 18 (24%) 0.038
Left colon 67 (34%) 34 (46%)
Rectum 51 (25%) 22 (30%)
Preoperative CEA levels (ng/mL), median (range) 3.8 (0.6–97.1) 4.1 (1.2–263.5) 0.182
Postoperative CEA levels (ng/mL), median (range) 2.6 (0.5–84.9) 3.1 (0.5–12.5) 0.009
T Stage
T1 20 (10%) 6 (8%) 0.912
T2 31 (15%) 10 (14%)
T3 139 (70%) 54 (73%)
T4 9 (5%) 4 (5%)
N Stage
N0 121 (61%) 48 (65%) 0.753
N1 56 (28%) 17 (23%)
N2 22 (11%) 9 (12%)
TNM stage
I 45 (23%) 14 (19%) 0.502
II 76 (38%) 34 (46%)
III 78 (39%) 26 (35%)
Differentiation
Well 6 (3%) 4 (6%) 0.490
Moderately 187 (94%) 69 (93%)
Poorly 6 (3%) 1 (1%)
LVI
Yes 119 (60%) 44 (59%) 0.959
No 80 (40%) 30 (41%)
PNI
Yes 23 (12%) 12 (16%) 0.307
No 176 (88%) 62 (84%)
Chemotherapy regimen
FOLFOX 77 (39%) 29 (39%) 0.843
XELOX 2 (1%) 0 (0%)
UFUR 52 (26%) 20 (27%)
Capecitabine 2 (1%) 0 (0%)
No 66 (33%) 25 (34%)
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Data are presented as n (%), unless otherwise indicated.

CEA, carcinoembryonic antigen; PNI, perineural invasion; LVI, lymphovascular invasion; TNM, tumor-node-metastasis

Disease-free survival rates

The overall 3-year DFS rate was significantly higher in group A (84.2%), with respect to group B (74.3%) and C (62.0%) (p = 0.001; Fig 1A). Among NS, the 3-year DFS rate was also significantly higher in group A with respect to group B and C (84.9% vs. 75.4% vs. 43.9%, p < 0.001; Fig 1B). However, among S, differences in the 3 year-DFS rates among groups A, B, and C diminished (82.3% vs. 69.2% vs. 83.3%, p = 0.772; Fig 1C).

Fig 1. Disease-free survival rates according to the CEA status.

Fig 1

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(a) Overall disease-free survival (DFS) rates. (b) Never/former smoker DFS rates. (c) Current smoker DFS rates. CEA, carcinoembryonic antigen.

The overall 3-year DFS rate was similar between smokers and nonsmokers (80.2% vs. 77.1%, p = 0.485; Fig 2A). In the subgroup analysis, the 3-year DFS rate was also similar between smokers and nonsmokers in groups A and B (78.8% vs. 81.8%, p = 0.853; Fig 2B). In contrast, smokers had higher 3 year-DFS rate than nonsmokers in group C (83.3% vs. 43.9%, p = 0.029; Fig 2C).

Fig 2. Disease-free survival rates between smokers and nonsmokers.

Fig 2

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(a) Overall disease-free survival (DFS) rates. (b) Patients with normal postoperative serum CEA levels. (c) Patients with elevated postoperative serum CEA levels. CEA, carcinoembryonic antigen.

Since the sample size of those on XELOX and capecitabine was too small for conclusions, we compared FOLFOX/XELOX versus UFUR/capecitabine instead. The analysis comparing between patients treated with different chemotherapy regimens showed no significant difference in DFS (FOLFOX/XELOX vs. UFUR/Capecitabine, 76.3% vs. 73.4%, p = 0.506).

Univariate analysis showed that preoperative serum CEA levels, postoperative serum CEA levels, tumor stage, age, lymphovascular invasion, and perineural invasion status were predictive of DFS (Table 3). In a multivariable analysis, only postoperative serum CEA levels, tumor stage, and age were significant independent prognostic factors for DFS (Table 4).

Table 3. Univariate analysis of prognostic factors for disease-free survival.

No. of patients 3-year DFS rate p
Sex
Male 149 74.5% 0.523
Female 124 81.0%
Age, years
< 75 168 81.2% 0.005
≥ 75 105 72.8%
Preoperative CEA
< 5 152 84.2% 0.002
≥ 5 121 70.1%
Postoperative CEA
< 5 221 81.1% < 0.001
≥ 5 52 62.0%
Tumor location
Right 99 76.2% 0.455
Left 101 74.0%
Rectum 73 83.7%
Differentiation
Well 10 67.5% 0.710
Moderately 256 78.6%
Poorly 7 71.4%
LVI
Yes 163 71.9% 0.023
No 110 85.9%
PNI
Yes 35 60.4% 0.010
No 238 80.5%
TNM Stage
I 59 93.7% < 0.001
II 110 84.4%
III 104 62.3%
Smoking
Yes 79 80.2% 0.485
No 194 77.1%
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CEA, carcinoembryonic antigen; PNI, perineural invasion; LVI, lymphovascular invasion; TNM, tumor-node-metastasis

Table 4. Multivariate analysis of prognostic factors for disease-free survival.

Hazard ratio 95% CI p
Postoperative CEA
< 5 1
≥ 5 1.814 1.016–3.241 0.044
TNM stage
I 1
II 2.085 0.797–5.457 0.135
III 4.603 1.791–11.828 0.002
Age, years
< 75 1
≥ 75 1.643 1.057–2.553 0.028
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CEA, carcinoembryonic antigen; TNM, tumor-node-metastasis

Optimal cutoff values of serum CEA levels for smoking patients

The optimal CEA level cutoff values for smoking patients were determined using ROC curve analysis. Preoperative and postoperative serum CEA levels showed no prognostic efficacy for colorectal cancer in smoking patients, with areas under the receiver operating characteristic curve (AUC) values of 0.543 and 0.583, respectively. The optimal cutoff value for preoperative serum CEA levels was 2.9 ng/ml; this had a sensitivity, specificity, and Youden index of 78.9%, 34.6%, and 0.136, respectively. Meanwhile, the optimal cutoff value for postoperative serum CEA levels was 2.2 ng/ml; this had a sensitivity, specificity, and Youden index of 85.0%, 31.4%, and 0.165, respectively.

Discussion

In this study, serum CEA level status significantly influenced the DFS rate of colon cancer patients. Patients with normal pre- and postoperative serum CEA levels (group A) had a better prognosis than did patients with elevated preoperative but normal postoperative CEA levels (group B). Patients with elevated postoperative serum CEA levels (group C) had the worst outcome. These results are consistent with those of previous studies [18,19]. However, as a novel finding, we show that current smokers with elevated postoperative CEA levels might not have a worse prognosis than non-smoking patients with similar postoperative CEA levels. To our best knowledge, this is the first study to determine the prognostic value of CEA status in smoking and nonsmoking patients with colorectal cancer.

Tobacco use is a well-known risk factor for serum CEA levels elevation [13–15]. Potential mechanisms by which smoking might increase CEA levels include chronic immune cell recruitment and inflammation [20]. Lung function and CEA levels significantly improve after 3 months of smoke cessation [21]. Thus, we placed former smokers (i.e., those who had quit smoking before or after surgery) in group NS along with never smokers, rather than in the S group, which consisted of patients who smoked both before and after surgery. Group S had higher postoperative CEA levels than did the NS group (2.6 vs. 3.1, p = 0.009), whereas both groups had similar preoperative levels (3.8 vs. 4.1, p = 0.182). This suggests that the contribution of cancer cells to serum CEA levels may be significantly higher than that of smoking in the preoperative period. Moreover, serum CEA values obtained in smoking patients may not reflect the actual levels of CEA produced by cancer cells, especially after surgery. This could explain why elevated postoperative CEA levels are less prognostic in current smokers.

Although smoking influences serum CEA concentrations, our data showed no significant differences in DFS between current smokers and former/never smokers (80.2% vs. 77.1%, p = 0.485). In our study, the smoker group included significantly younger patients (median age, 73 vs. 68 years, p = 0.009), which may have affected this result. The association between smoking status and survival in patients with colorectal cancer has yet to be established, with most [22–25] but not all studies [26–28], linking cigarette smoking to worse survival rates.

Serum CEA level is a widely accepted tumor marker, particularly for colorectal cancer, and its determination is standardized, inexpensive, and easily available. Elevated preoperative CEA levels are thought to be an independent prognostic factor in colorectal carcinoma. In previous studies, overall survival rates were lower when preoperative serum CEA levels were elevated, regardless of the disease stage [3,19,29]. However, some studies reported that postoperative serum CEA levels were better predictors than were preoperative serum CEA levels. In these studies, postoperative, but not preoperative, CEA status was a significant prognostic predictor in multivariable analyses [7,9,30]. In fact, current guidelines do not support the use of elevated preoperative CEA as an indicator for adjuvant chemotherapy [1,2]. Konishi el al. found no significant differences in the 3-year DFS rates between patients with normal postoperative serum CEA levels and those with normal preoperative CEA levels [9]. Elevated preoperative serum CEA levels are not informative when the postoperative levels are normal; thus, preoperative serum CEA level determination could be disregarded as a prognostic factor. In agreement, elevated postoperative serum CEA levels were a better predictor than elevated preoperative serum CEA levels in our multivariable model.

In our study, preoperative and postoperative serum CEA levels showed no prognostic efficacy for smoker patients, with AUC values close to 0.5 (0.543 and 0.583, respectively). It was not possible to redefine serum CEA level elevation by simply increasing the threshold because we found no linear correlation between serum CEA values and DFS in this subgroup. The optimal cutoff values for preoperative and postoperative serum CEA levels in smokers were, respectively, 2.9 ng/ml and 2.2 ng/ml, which were even lower than the global standard CEA cutoff value of 5.0 ng/ml. As such, we failed to identify a predictive threshold for smokers before and after surgery.

In addition to CEA status, we found that tumor stage and age were also independent prognostic factors for DFS on multivariable analysis. Regional lymph node involvement is one of the strongest predictors of outcome following surgical resection of colorectal cancers. Nodal spread, rather than elevated serum CEA concentrations, is an indication for adjuvant therapy for colorectal cancer in most guidelines [1,2]. Advanced age has been shown to reduce overall survival and DFS rates and, to a lesser extent, cancer-specific survival rates in patients with colorectal cancer [31–34].

The major limitation of our study is the variability of the adjuvant chemotherapy regimens. However, an analysis comparing the four chemotherapy regimens (XELOX, FOLFOX, oral UFUR, and oral capecitabine) showed no significant differences in DFS among them. As a further limitation, we quantified smoking only according to self-reports at a single time point before surgery, a method that is relatively unreliable. Additional limitations include the relatively small number of former smokers in the NS group, which likely resulted in limited statistical power, and the lack of consideration of factors (e.g., diabetes, liver disease, and acute or chronic inflammation) that can also generate confusing CEA results. Under consideration of cost, molecular tests were only reserved for patients with stage IV disease in our hospital. The patients included in this study lacked molecular profiles such as KRAS, NRAS, and BRAF. Lastly, our study was retrospective, with a relatively small number of patients, and the follow-up period for some patients was short. In the future, large prospective studies analyzing CEA kinetics via measurements of follow-up serum CEA levels are required. The amount and duration of smoking and type of cigarette should also be taken into consideration in future studies.

Conclusion

Our findings demonstrate that persistent smoking can increase serum CEA levels in patients with colorectal cancer, affecting the postoperative prognostic value of serum CEA levels in current smokers. Elevated postoperative serum CEA level is associated with lower DFS rates in never and former smokers, but not in current smokers. Therefore, colorectal cancer patients that are current smokers may need the characterization of alternative tumor markers, useful as surveillance strategy.

Supporting information

S1 Raw Data

(XLSX)

Click here for additional data file. (467.1KB, xlsx)

Acknowledgments

The authors would like to acknowledge Wei-Ching Lin for her help in coordinating the clinical aspects of this study.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

National Yang-Ming University Hospital The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Jason Chia-Hsun Hsieh

30 Mar 2020

PONE-D-20-04276

Prognostic value of serum carcinoembryonic antigen levels in colorectal cancer patients who smoke

PLOS ONE

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Reviewer #2: No

Reviewer #3: Yes

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Reviewer #1: In this study, authors aimed to evaluate the impact of smoking on the prognostic value of serum CEA levels. In this single site, they retrospectively collected clinical data, treatment outcomes, smoking status and CEA level before and after operation. They found that although patients whose CEA levels were normal preoperative and postoperative showed the best 3-year DFS, elevated postoperative CEA levels were associated with lower DFS rates in never and former smokers, but were not associated with lower DFS rates in current smokers.

The strength of this study is that they found that persistent smoking altered the prognostic value of postoperative serum CEA levels in colorectal cancer patients. This is the first study to determine the prognostic value of CEA status in smoking and nonsmoking patients with colorectal cancer. Before accepted for publication, they are still some issues.

Major issue

1. Different stage will lead to different outcome. In colon cancer, molecular markers such as K-Ras,N-Ras and B-Raf are also an important factors. This study included stage I, II and III colon-rectal cancer. Authors should list the distribution of stages within different groups. If possible, analysis of molecular subtypes are also important.

2. In line 223, authors mentioned about groups C had the worse outcome. But in group C, there were more patients with right side tumors compared with Group B and A, and more patients were LN positive compared with Group A. In summery, group C was already with worse prognostic factors, nothing to do with CEA level before and after operation. Interesting, fewer patients in this group had received adjuvant chemotherapy compared with group B, which might also lead to worse outcome.

Minor issues

1. Authors may add regimen of different adjuvant chemotherapy in each group.

2. How can authors distinguish local recurrence from metachronous colon cancers (line 149)?

3. In line 280, authors mentioned an analysis comparing the four chemotherapy regimens showed no significant differences in DFS among them. Can you provide the reference?

4. In this study, authors should define current smoker and former smoker more accurately. You can see a paper which was published at J Clin Oncol. 2013 Jun 1;31(16):2016-23. Especially former smoker, some patients might just stop smoking before operation. Another issue is previous smoking might already lead to lung injury, which may also lead to elevation of CEA.

Reviewer #2: According to your statistic method, I don’t think it support your conclusion and the title. You should focus on the patients with persistent elevated CEA and using NS/S to compare disease-free survival if prognostic factors are balanced between two groups.

Reviewer #3: General Comments:

This is a generally well-written manuscript.

However, there are still some issues regarding scientific novelty, scientific interpretation of the results that need to be well-addressed.

Major comments:

1. In group C, the author defined elevated pre-operative and post-operative CEA group. However, in table1, the range of pre-operative CEA level is 2.8-263.5 ng/ml. Which means some patient’s CEA level was normal before operation.

The author should carefully defined the group C.

2. One important finding in this study is post-operative CEA level is significantly higher in smoking group than non-smoking group (2.6 vs. 3.1 ng/ml). Although the p value is significant, the difference is too small in clinical practice. My other question is in non-smoking group, the mean post-operative CEA level is 2.6 ng/ml, range from 0.5-84.9 ng/ml. However, in smoking group, the mean post-operative CEA level is 3.1 ng/ml, range from 0.5-12.5 ng/ml. It seems the distribution of CEA level in non-smoking group is very wide. Do the author have the confidence to claim the post-operative CEA level is really higher in smoking group than non-smoking group?

Minor comments:

In abstract, the post-operative CEA level in smoking group is 2.5 ng/ml. However, in the table 2, the level is 2.6 ng/ml.

**********

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Reviewer #2: No

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PLoS One. 2020 Jun 5;15(6):e0233687. doi: 10.1371/journal.pone.0233687.r002

Author response to Decision Letter 0

13 Apr 2020

Reviewer #1

Comments 1:

Different stage will lead to different outcome. In colon cancer, molecular markers such as K-Ras,N-Ras and B-Raf are also an important factors. This study included stage I, II and III colon-rectal cancer. Authors should list the distribution of stages within different groups. If possible, analysis of molecular subtypes are also important.

Response 1:

Yes. We completely agree with the reviewer’s pertinent comments. The distribution of stages within different groups are now provided in the table below and in the revised manuscript (Results section, Table 1, page 9).

Group A

(n = 152) Group B

(n = 69) Group C

(n = 52) p

TNM stage

I 47 (31%) 5 (7%) 7 (13%) < 0.001

II 62 (41%) 29 (42%) 19 (37%)

III 43 (28%) 35 (51%) 26 (50%)

We agree that the molecular markers play an important role in colorectal cancer prognosis. However, under consideration of cost, molecular tests were only reserved for patients with stage IV disease in our hospital. The patients included in this study lacked molecular profiles such as KRAS, NRAS, and BRAF. We added this as a limitation in the revised manuscript (Discussion section, page 19, lines 295-297).

Comments 2:

2. In line 223, authors mentioned about groups C had the worse outcome. But in group C, there were more patients with right side tumors compared with Group B and A, and more patients were LN positive compared with Group A. In summery, group C was already with worse prognostic factors, nothing to do with CEA level before and after operation. Interesting, fewer patients in this group had received adjuvant chemotherapy compared with group B, which might also lead to worse outcome.

Response 2:

Yes. We agree with the reviewer’s insightful concern. Tumor sides, tumor stage, and chemotherapy are all well-known prognostic factors of colorectal cancer. As shown in Fig 1a, we found that in the CEA group, the overall 3-year DFS rate was significantly higher in group A (84.2%), with respect to group B (74.3%) and C (62.0%). Univariate analysis in our study showed that the CEA group was predictive of disease-free survival.

Univariate analysis of the CEA group for disease-free survival

No. of patients 3-year DFS rate p

CEA group

Group A 152 84.2% 0.001

Group B 69 74.3%

Group C 52 64.7%

If we select the CEA group rather than the preoperative and postoperative CEA as a variable, CEA group, tumor stage, and age were significant independent prognostic factors for disease-free survival in multivariable analysis.

Multivariate analysis of prognostic factors for disease-free survival

Hazard ratio 95% CI p

CEA group

Group A 1

Group B 1.299 0.760–2.222 0.338

Group C 2.356 1.369–4.060 0.002

TNM stage

I 1

II 2.085 0.797–5.457 0.135

III 4.603 1.791–11.828 0.002

Age, years

< 75 1

≥ 75 1.643 1.057–2.553 0.028

As a result, we believe that tumor side, stage, chemotherapy, and elevated postoperative CEA may all have contributed to the poorer prognosis in group C.

Comments 3:

Authors may add regimen of different adjuvant chemotherapy in each group.

Response 3:

We thank the reviewer for the insightful comment. The regimen of different adjuvant chemotherapy within different groups are provided in the tables below and in the revised manuscript

Regimen of adjuvant chemotherapy within CEA groups

Group A

(n = 152) Group B

(n = 69) Group C

(n = 52) p

Regimen

FOLFOX 54 (36%) 36 (52%) 16 (31%) < 0.001

XELOX 0 (0%) 2 (3%) 0 (0%)

UFUR 35 (23%) 19 (28%) 18 (34%)

Capecitabine 0 (0%) 0 (0%) 2 (4%)

No 63 (41%) 12 (17%) 16 (31%)

(Results section, Table 1, page 9)

Regimen of adjuvant chemotherapy within smoking groups

NS (n = 199) S (n = 74) p

Regimen

FOLFOX 77 (39%) 29 (39%) 0.843

XELOX 2 (1%) 0 (0%)

UFUR 52 (26%) 20 (27%)

Capecitabine 2 (1%) 0 (0%)

No 66 (33%) 25 (34%)

(Results section, Table 2, page 12)

Comments 4:

How can authors distinguish local recurrence from metachronous colon cancers?

Response 4:

We thank the reviewer for the thoughtful comment. Local recurrence was observed in 13 patients in our study. Most of these cases initially had locally advanced cancer with adjacent organ involvement. The clinical features of patients with local recurrence are shown in the table below.

Case No. Recurrent Site Synchronous Metastasis

1 Pelvis Nil

2 Pelvis Liver

3 Abdominal wall Paraaortic lymph nodes

4 Pelvis Liver

5 Pelvis Liver

6 Abdominal wall Nil

7 Psoas muscle Liver

8 Abdominal wall Nil

9 Anastomosis Ovary

10 Bladder Nil

11 Anastomosis Lung

12 Abdominal wall Nil

13 Abdominal wall Carcinomatosis

Based on the clinical features we presented above, these cases could be distinguished from metachronous colon cancer.

Comments 5:

In line 280, authors mentioned an analysis comparing the four chemotherapy regimens showed no significant differences in DFS among them. Can you provide the reference?

Response 5:

Yes. We thank the reviewer for the insightful comments. Univariate analysis of chemotherapy regimens for disease-free survival are provided in the table below and in the revised manuscript

Univariate analysis of chemotherapy regimens for disease-free survival

No. of patients 3-year DFS rate p

Regimen

FOLFOX / XELOX 108 76.3% 0.506

UFUR / Capecitabine 74 73.4%

The sample sizes of those on XELOX and capecitabine were too small for conclusions. As a result, we compared between FOLFOX/XELOX versus UFUR/Capecitabine instead. (Results section, page 13, lines 203-206)

Comments 6:

In this study, authors should define current smoker and former smoker more accurately. You can see a paper which was published at J Clin Oncol. 2013 Jun 1;31(16):2016-23. Especially former smoker, some patients might just stop smoking before operation. Another issue is previous smoking might already lead to lung injury, which may also lead to elevation of CEA.

Response 6:

Yes. We agree with the reviewer’s astute concern, some patients might just have stopped smoking before the operation, and previous smoking might have already led to lung injury. However, the lung function and CEA levels could significantly improve after 3 months of smoke cessation [1]. Thus, we placed former smokers in the group NS along with never smokers, rather than in the S group, which included patients who smoked both before and after the surgery.

We agree that CEA levels are supposed to be different between never smokers and former smokers. However, one of the limitations of our study include the relatively small number of former smokers (n = 26), which likely resulted in limited statistical power. The amount and duration of smoking and type of cigarette would be taken into consideration in our future studies.

1. Pezzuto A, Spoto C, Vincenzi B, Tonini G. Short-term effectiveness of smoking-cessation treatment on respiratory function and CEA level. J Comp Eff Res. 2013;2: 335-343.

Reviewer #2

Comments 1:

According to your statistic method, I don’t think it support your conclusion and the title. You should focus on the patients with persistent elevated CEA and using NS/S to compare disease-free survival if prognostic factors are balanced between two groups.

Response 1:

Yes. We thank the reviewer for the thoughtful comments. We did compare disease-free survival between smoker and nonsmoker patients with persistent elevated CEA (Results section page 12, in line 189). In subgroup analysis, smokers had higher 3 year-DFS rates than nonsmokers in group C (83.3% vs. 43.9%, p = 0.029; Figure 2).

According to your suggestion, we have revised our title for the manuscript. Further, we also added more descriptions and two figures below and in the revised manuscript to demonstrate the 3 year-DFS rates between smokers and nonsmokers in different subgroups.

“The overall 3-year DFS rate was similar between smokers and nonsmokers (80.2% vs. 77.1%, p = 0.485; Fig 2a). In the subgroup analysis, the 3-year DFS rate was also similar between smokers and nonsmokers in groups A and B (78.8% vs. 81.8%, p = 0.853; Fig 2b). In contrast, smokers had higher 3 year-DFS rate than nonsmokers in group C (83.3% vs. 43.9%, p = 0.029; Fig 2c).” (Results section, pages 12-13, lines 189-193)

Fig 2. Disease-free survival rates between smokers and nonsmokers (a) Overall disease-free survival (DFS) rates. (b) patients with normal postoperative serum CEA levels. (c) patients with elevated postoperative serum CEA levels.

Reviewer #3

Comments 1:

In group C, the author defined elevated pre-operative and post-operative CEA group. However, in table1, the range of pre-operative CEA level is 2.8-263.5 ng/ml. Which means some patient’s CEA level was normal before operation. The author should carefully defined the group C.

Response 1:

Yes. We thank the reviewer for the thoughtful comments. There were 6 patients with normal preoperative CEA and elevated postoperative CEA in group C. One of them had tumor recurrence as carcinomatosis, 10 months after curative surgery. The clinical features of the patients are shown in the table below.

Case No. Preoperative CEA Postoperative CEA Recurrence

1 4.8 7.4 Carcinomatosis

2 4 6.1 Nil

3 2.8 6.5 Nil

4 3.7 9.3 Nil

5 3.9 6.4 Nil

6 4.1 5.1 Nil

According to your suggestion, we have redefined group C as patients with elevated postoperative CEA in the revised manuscript.

Comments 2:

One important finding in this study is post-operative CEA level is significantly higher in smoking group than non-smoking group (2.6 vs. 3.1 ng/ml). Although the p value is significant, the difference is too small in clinical practice. My other question is in non-smoking group, the mean post-operative CEA level is 2.6 ng/ml, range from 0.5-84.9 ng/ml. However, in smoking group, the mean post-operative CEA level is 3.1 ng/ml, range from 0.5-12.5 ng/ml. It seems the distribution of CEA level in non-smoking group is very wide. Do the author have the confidence to claim the post-operative CEA level is really higher in smoking group than non-smoking group?

Response 2:

Yes. We thank the reviewer for the thoughtful comments. We have used Mann-Whitney U test to compare the postoperative CEA between smokers and nonsmokers. Although there are few mild outliers and extreme outliers among the nonsmoker group, the difference in the median postoperative CEA between smokers and nonsmokers was statistically significant (p = 0.009). The distribution of postoperative CEA is shown in the box and whisker plot below.

Although the absolute median difference between the two groups is relatively small, the proportion of patients with elevated postoperative CEA is significantly higher in the smoker group (32% vs. 14%, p <0.001). However, smokers with elevated postoperative CEA levels had a higher 3 year-DFS rates than non-smoking patients with similar postoperative CEA levels (83.3% vs. 43.9%, p = 0.029). These findings support our conclusion that postoperative serum CEA levels showed no prognostic efficacy for smoker patients

Comments 3:

In abstract, the post-operative CEA level in smoking group is 2.5 ng/ml. However, in the table 2, the level is 2.6 ng/ml.

Response 3:

Yes, we thank the reviewer for the kind reminder. We have corrected the abstract data in the revised manuscript.

Attachment

Submitted filename: Response_to_Reviewers.docx

Click here for additional data file. (341.2KB, docx)

Decision Letter 1

Jason Chia-Hsun Hsieh

12 May 2020

Prognostic value of postoperative serum carcinoembryonic antigen levels in colorectal cancer patients who smoke

PONE-D-20-04276R1

Dear Dr. Huang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Jason Chia-Hsun Hsieh, M.D. Ph.D

Academic Editor

PLOS ONE

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All the questions were answered adequately.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: (No Response)

**********

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Acceptance letter

Jason Chia-Hsun Hsieh

21 May 2020

PONE-D-20-04276R1

Prognostic value of postoperative serum carcinoembryonic antigen levels in colorectal cancer patients who smoke

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