**About the Editor**

#### **Ugo Grossi**

Dr. Ugo Grossi is an accomplished medical professional with a distinguished educational background and a wealth of experience in the field of gastrointestinal surgery. He earned his Medical Degree from the Catholic University of Rome in 2007, followed by a Postgraduate Degree in Surgery from the same institution at A. Gemelli University Hospital IRCCS in 2015. Dr. Grossi furthered his academic journey by completing a PhD in Digestive Diseases at Queen Mary University of London, UK, in 2021. Dr. Grossi has been an Assistant Professor at the University of Padua since 2022, and his research interests are primarily centered around the surgical management of benign coloproctological conditions, including functional and inflammatory bowel disorders, as well as pelvic floor disorders. His extensive contributions to this field are evident through his numerous publications. Notably, he has been recognized for his outstanding work with awards from esteemed organizations such as the Italian Society of Surgery (Ettore Ruggieri Award), the European Crohn's and Colitis Organisation (ECCO Travel Award), and the Italian Society of Colorectal Surgery (John Nicholls Prize). His dedication to advancing the field is further underscored by his recent appointment to the Anorectal Disorders Committee for the V Revision of the Rome Criteria for the Rome Foundation.

## *Article* **Association of Albumin-Corrected Serum Calcium Levels with Colorectal Cancer Survival Outcomes**

**Marina Nogueira Silveira, Lara Pozzuto, Maria Carolina Santos Mendes, Lorena Pires da Cunha, Felipe Osório Costa, Lígia Traldi Macedo, Sandra Regina Brambilla and José Barreto Campello Carvalheira \***

> Division of Oncology, Department of Anesthesiology, Oncology and Radiology, School of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP 13083-888, Brazil; marina.nogueira2@gmail.com (M.N.S.); larapozzuto.nutri@gmail.com (L.P.); mariacarol.op@gmail.com (M.C.S.M.); lorenapcunha@yahoo.com (L.P.d.C.); felipeoc@unicamp.br (F.O.C.); ligiamed@gmail.com (L.T.M.); sandraunicamp2@gmail.com (S.R.B.)

**\*** Correspondence: jbcc@unicamp.br; Tel.: +55-19-3521-7496

**Abstract:** In epidemiological studies, higher calcium intake has been associated with decreased colorectal cancer (CRC) incidence. However, whether circulating calcium concentrations are associated with CRC prognosis is largely unknown. In this retrospective cohort analysis, we identified 498 patients diagnosed with stage I–IV CRC between the years of 2000 and 2018 in whom calcium and albumin level measurements within 3 months of diagnosis had been taken. We used the Kaplan– Meier method for survival analysis. We used multivariate Cox proportional hazards regression to identify associations between corrected calcium levels and CRC survival outcomes. Corrected calcium levels in the highest tertile were associated with significantly lower progression-free survival rates (hazard ratio (HR) 1.85; 95% confidence interval (CI) 1.28–2.69; *p* = 0.001) and overall survival (HR 1.86; 95% CI 1.26–2.74, *p* = 0.002) in patients with stage IV or recurrent CRC, and significantly lower disease-free survival rates (HR 1.44; 95% confidence interval (CI) 1.02–2.03; *p* = 0.040) and overall survival rates (HR 1.72; 95% CI 1.18–2.50; *p* = 0.004) in patients with stage I–III disease. In conclusion, higher corrected calcium levels after the diagnosis of CRC were significantly associated with decreased survival rates. Prospective trials are necessary to confirm this association.

**Keywords:** gastrointestinal malignancies; cancer survivorship; calcium carbonate; hypercalcemia; cancer outcomes

#### **1. Introduction**

Every year, it is expected that approximately 1.88 million people will be diagnosed with colorectal cancer (CRC) worldwide, while 915,880 deaths are attributed to the disease [1]. Although current changes in risk factors such as decreased smoking and red meat consumption may have contributed to the decline in overall incidence of CRC in some countries, it is still the third most common type of cancer [2]. For this reason, research that considers possible predictive as well as prognostic factors is needed.

Higher calcium intake has been associated with a decreased risk of CRC [3–5]. Results from a meta-analysis of 21 publications showed that for each 300 mg of calcium consumed, there was a reduction of 8–9% in the risk of acquiring CRC [6]. Consistently, a pooled analysis of 534,536 individuals also revealed a reduction in CRC with higher calcium intake [7]. In contrast, a recently published, large prospective trial found that calcium and calcitriol supplementation was associated with an increased risk of the development of serrated polyps, a CRC precursor lesion [8]. Although calcium signaling is a key player in the fundamental stages of cancer development, the complexities of calcium intersections with oncogenic pathways are context dependent (i.e., the alignment of calcium channels in cancer cells, extracellular calcium concentrations, and calcium interactions with the microenvironment are factors that determine calcium influence in cancer cell fate) [9]. Moreover, increased levels of extracellular calcium are insufficient to modulate cancer

**Citation:** Silveira, M.N.; Pozzuto, L.; Mendes, M.C.S.; da Cunha, L.P.; Costa, F.O.; Macedo, L.T.; Brambilla, S.R.; Carvalheira, J.B.C. Association of Albumin-Corrected Serum Calcium Levels with Colorectal Cancer Survival Outcomes. *J. Clin. Med.* **2022**, *11*, 2928. https://doi.org/ 10.3390/jcm11102928

Academic Editors: Jaydutt V. Vadgama and Ugo Grossi

Received: 14 April 2022 Accepted: 19 May 2022 Published: 22 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

cell proliferation. Rather, cytosolic calcium levels, which are mainly determined by the activity of calcium channels, pumps, and exchangers, are key to the control of intracellular calcium levels in a context-dependent manner [9]. Thus, it is simple to delineate a plausible biological framework of how the modulation of extracellular calcium levels by calcium intake influences carcinogenesis.

Much less is known about the effects of serum calcium on the risk of developing CRC. In striking opposition to the association between calcium intake and a reduced risk of CRC, a retrospective analysis of a Swedish databank (Apolipoprotein Mortality Risk (AMORIS)) showed a modest increase in the risk of developing CRC in the highest quartiles of albumin-corrected serum calcium [10]. Calcium homeostasis is regulated not only by calcium intake but also by bidirectional fluxes of this ion at the level of the kidneys and bones [11]. Importantly, calcium homeostasis is disrupted in 20–30% of patients during the course of cancer development [12], making it biologically plausible that the association between higher calcium levels and CRC observed in the AMORIS study may be related to calcium homeostasis disturbances mediated by the initial stages of tumor development.

In normal physiology, extracellular calcium levels are mainly regulated by parathyroid hormone (PTH), calcitonin, and calcitriol [11]. On the other hand, in cancer pathology, the vast majority of hypercalcemia of malignancy is associated with increased PTH-related peptide (PTHrP) levels [12]. Interestingly, PTHrP has a limited role during cartilage embryogenesis through the activation of the hedgehog signaling pathway in cartilage cells [13,14]. In cancer cells, this dormant pathway is reactivated, increasing circulating levels of PTHrP [15], which leads to the elevation of the extracellular calcium concentration in a tumor-burden- and aggressiveness-dependent manner [16,17]. Thus, bearing in mind the hypothesis that the corrected calcium level is a biomarker of cancer progression, we sought to examine the impact of corrected calcium levels in the outcomes of patients with CRC, using both calcium and albumin levels routinely measured during clinic visits reported in medical records.

#### **2. Materials and Methods**

#### *2.1. Study Population*

This study was a single-center, retrospective, and analytical study conducted at the State University of Campinas Hospital (HC-UNICAMP) in Campinas, Brazil. The study population was composed of patients diagnosed with stage I–IV CRC between the years of 2000 and 2018, admitted to the HC-UNICAMP. Patients that met the following inclusion criteria were selected: histologically confirmed CRC between 2000 and 2018; CRC stage I–IV according to the 8th edition of the American Joint Committee on Cancer (AJCC) cancer staging manual [18]; the availability of calcium and albumin measurements within 3 months of the diagnosis for stage IV or recurrent CRC; the availability of calcium and albumin measurements before surgery in patients with stage I–III CRC; and complete medical record information regarding age, date of diagnosis, topography, histological type, and tumor staging. Patients with concomitant malignancies, CRC that was not adenocarcinoma, in situ CRC, or unreported data regarding treatment were excluded (Figure 1).

The study was approved by the local Institutional Review Board (CAAE number: 15505419.1.0000.5404) with a consent form waiver. The principles recommended by the Declaration of Helsinki were adhered to.

#### *2.2. Body Composition*

Two consecutive computed tomography (CT) images of the third lumbar vertebra were evaluated; the images were obtained from routine examinations of the patients. Baseline imaging was performed within 3 months of diagnosis for patients with stage I–III CRC and 3 months before diagnosis or chemotherapy initiation for patients with stage IV or recurrent CRC. Skeletal muscle (SM) values of the psoas, abdominal, rectus abdominis, and paravertebral muscles were measured [19,20]. The visceral adipose tissue (VAT), intramuscular adipose tissue (IMAT), and subcutaneous adipose tissue (SAT) were also

measured; from these values, we determined the SM index (SMI), the SAT index (SFI), and the VAT index (VFI), measured in units of square centimeters (cm2) and normalized by height in square meters (m2). The software used was SliceOmatic V. 5.0. (Tomovision, Canada); standard Hounsfield units (HUs) established for tissues were −150 to −50 for VAT, −190 to −30 for IMAT and SAT, and −29 to 150 for SM. The images were analyzed by two evaluators (M.N.S. and L.P.) blinded to the outcomes, and the coefficients of variation for the cross-sectional areas analyzed were 1.07%, 1.05%, 1.61%, and 3.57% for SM, SAT, VAT, and IMAT, respectively, and 1.60% for SM density.

**Figure 1.** Study flowchart.

#### *2.3. Data Collection*

Data were collected from medical records, specifically from the time of CRC diagnosis until the date of death or last follow-up. Research Electronic Data Capture (REDCap) software was used for the construction of case report forms (CRFs) and database management [21].

#### 2.3.1. Clinical Variables

The variables collected comprised sociodemographic characteristics (age, sex, ethnicity, smoking status, and alcohol use status) and anthropometric characteristics (weight, weight loss (WL), height, and body mass index (BMI) at diagnosis). Additionally, disease-related covariates were obtained regarding the date of CRC diagnosis, the Eastern Cooperative Oncology Group Performance Status Scale (ECOG) status, chemotherapy regimens, the primary tumor location, the Charlson Comorbidity Index [22], carcinoembryonic antigen (CEA), the number of metastases, emergency surgery, and the clinical and pathological stage according to the AJCC cancer staging manual (tumor, node, and metastasis (TNM)) [23].

#### 2.3.2. Biochemical Exam Data

Serum albumin and calcium levels were measured using calorimetric assays according to the HC-UNICAMP clinical pathology protocol. Calcium (mg/dL; reference range: 8.8–10.2 mg/dL for adults aged 21–50 years and 8.4–9.7 mg/dL for adults > 50 years old), albumin (mg/dL; reference range: 3.4–4.8 g/dL), baseline CEA levels (ng/mL; cut-off value: 5 ng/mL), and complete blood count levels were collected within 3 months of diagnosis for patients with stage IV or recurrent CRC, and before surgery for patients with stage I–III CRC.

#### 2.3.3. Corrected Calcium Measurement

Given that ionized calcium is not measured routinely in clinics, we used corrected calcium to estimate the free calcium concentration, which was calculated using the following formula: corrected calcium = serum calcium + [(4.0 − serum albumin) × 0.8] [24]. The corrected calcium levels were categorized into tertiles.

#### 2.3.4. Systemic Inflammatory Indexes

The neutrophil-to-lymphocyte ratio (NLR) was calculated by dividing the absolute count of neutrophils by the absolute count of lymphocytes [25]. The platelet-to-lymphocyte ratio (PLR) was calculated by dividing the absolute count of platelets by the absolute count of lymphocytes [26]. The lymphocyte-to-tomonocyte ratio (LMR) was calculated by dividing the absolute count of lymphocytes by the absolute count of monocytes. The NLR and PLR were analyzed as continuous variables.

#### 2.3.5. Endpoints

The co-primary endpoints were progression-free survival and overall survival, which were calculated using the time between disease diagnosis or recurrence and the first event (disease progression or death) and death from any cause, respectively. Data regarding mortality were obtained from medical records. To evaluate the outcomes, the last date of follow-up recorded in the medical record or the date of death of the patient was considered.

#### *2.4. Statistical Analysis*

After summarizing the baseline characteristics based on the corrected calcium levels using descriptive statistics, the characteristics were compared by using chi-square and Kruskal–Wallis tests. Multivariate-adjusted Cox proportional hazards regression models were used to investigate associations between corrected calcium and progression-free and overall survival. Time was calculated in months from the diagnosis to the time of the event or the last follow-up visit (through August 2018).

To minimize the effects of potential confounders in our regression model, we included variables related to CRC-specific mortality outcomes established in previous studies. We also included variables that were associated (*p* < 0.10 in the unadjusted Cox analysis) with CRC mortality. We used the Kaplan–Meier method for survival analysis.

Analyses stratified by stage, cancer site, age, and gender were performed. Two-sided *p* values < 0.05 were considered to be statistically significant. The STATA 12 software was used for statistical analysis.

#### **3. Results**

#### *3.1. Patient Disposition and Baseline Characteristics*

A total of 256 patients with stage IV or recurrent CRC were included in our study; 207 died of any cause, with a median follow-up of 15.7 months (interquartile range (IQR) 5.8–32.6 months) at the time of the analysis.

Baseline characteristics according to corrected calcium levels are shown in Table 1. Generally, subjects with high levels of corrected calcium (≥9.46 mg/dL) were younger, had more metastases, the highest CEA levels, were less often submitted to prior neoadjuvant or adjuvant treatment, and were less often submitted to a backbone chemotherapy regimen with oxaliplatin. The other characteristics evaluated were similar among the calcium levels.

We also evaluated 243 patients with CRC stage I–III for calcium levels < 9.44 mg/dL and ≥9.44 mg/dL; only sex correlated with higher calcium levels (*p* = 0.002) (Table S1).


**Table 1.** Selected demographic and clinical characteristics and laboratory findings according to calcium tertiles of patients with metastatic colorectal cancer.

Abbreviations: BMI: body mass index; CEA: carcinoembryonic antigen; ECOG: Eastern Cooperative Oncology Group Performance Scale; IQR: interquartile range. <sup>a</sup> Chi-square test, <sup>b</sup> Fisher's exact test, <sup>c</sup> Kruskal–Wallis test.

#### *3.2. Body Composition and Inflammatory Indexes*

The serum calcium levels of patients with stage IV or recurrent CRC showed no correlation with body composition variables; however, when evaluating inflammatory markers, there were higher levels of the NLR and the PLR (Table S2).

In non-metastatic patients, serum calcium levels ≥ 9.44 mg/dL correlated with lower IMAT (*p* = 0.016), lower NLR (*p* = 0.005), and higher LMR (*p* = 0.025) (Table S3).

#### *3.3. Survival Analysis*

As shown in Table 2, unadjusted Cox regression revealed that higher levels of corrected calcium were associated with reduced median progression-free survival (*p* < 0.001) and overall survival (*p* < 0.001) rates in patients with stage IV or recurrent CRC. The significant association persisted after adjusting the model for age, BMI, ECOG, baseline CEA levels, the number of metastases, chemotherapy use, and WL. High levels of calcium were associated with decreased median progression-free survival (hazard ratio (HR) 1.85; 95% confidence interval (CI) 1.27–2.69, *p* = 0.001) (Figure 2a) and overall survival rates (HR 1.86; 95% CI 1.26–2.74, *p* = 0.002) (Figure 2b).

**Table 2.** Corrected calcium and survival for patients with metastatic colorectal cancer.


Abbreviations: CI, confidence interval; HR, hazard ratio. <sup>a</sup> Cox model adjusted for age, body mass index, Eastern Cooperative Oncology Group Performance Scale, number of metastases, chemotherapy use, and weight loss.

Likewise, higher levels of calcium were associated with decreased progression-free survival (HR 1.44; 95% CI 1.02–2.03, *p* = 0.040) (Figure 3a) and overall survival rates (HR 1.72; 95% CI 1.18–2.50, *p* = 0.004) (Figure 3b) in patients with stage I–III CRC, even after adjusting the model for age, BMI, WL, smoking, the Charlson Comorbidity Index, cancer stage, and emergency surgery.

**Figure 3.** Survival curves of patients with stage I–III colorectal cancer divided by corrected calcium levels (in mg/dL): (**a**) disease-free survival and (**b**) overall survival.

#### **4. Discussion**

In this retrospective cohort, patients with metastatic disease presented with significantly decreased progression-free and overall survival rates in a corrected-calciumdependent manner. Importantly, this association appeared to be independent of age, BMI, ECOG, CEA levels, the number of metastases, chemotherapy regimen, and WL. Moreover, patients with non-metastatic disease had increased risk of progression and mortality even after adjusting the model for age, BMI, WL, smoking, the Charlson Comorbidity Index, and emergency surgery. However, our analysis with body composition was not associated with corrected calcium levels.

Previous studies have evaluated the effects of hypercalcemia of malignancy (calcium greater than upper limit of normal (ULN)) on disease outcomes but not the influence of calcium as a biomarker of CRC progression. These reports consistently associate hypercalcemia with a poor prognosis [27,28]. Thus, a key question is that beyond corrected calcium directly influencing cancer outcomes, is it also a biomarker of cancer progression?

Interestingly, a few reports have investigated the role of corrected calcium as a marker of cancer progression without categorizing it in the ULN. For example, in the setting of metastatic kidney cancer, the set level of corrected calcium commonly used in prognostic models of survival (a tool used routinely in clinics) is lower than the ULN [29,30]. Interestingly, extracellular calcium levels per se have an established CRC chemoprotective effect [31–33]. Extracellular calcium levels may be considered a marker for increased PTH and vitamin D levels. Like extracellular calcium levels, serum vitamin D level is associated with reduced cancer mortality [34]; thus, a possible biological explanation for the association of corrected calcium levels with cancer progression is that corrected calcium may reflect the spectrum of PTHrP secreted by the tumor. PTHrP is expressed in >90% of CRC cases, and the grade of its expression was previously correlated with poor differentiation and aggressiveness [17]. We also found an association between higher levels of corrected calcium and a greater number of metastases and higher levels of CEA, which are important prognostic biomarkers for metastatic CRC. Consistent with this finding, PTHrP was recently linked to the increased proliferation of colon cancer cells [35]. Moreover, elevated PTHrP levels have been associated with cancer cachexia [16]. However, in our analysis, body composition, excluding IMAT in nonmetastatic patients, was not associated with

corrected calcium, corroborating the findings of a recent study where the serum PTHrP level was not correlated with WL, uncoupling protein (UCP)-1, and other white adipose tissue browning markers [36]. Thus, additional prospective studies are needed to elucidate the role of PTHrP in determining body composition in humans.

Interestingly, in a cohort of patients with gastroesophageal cancer, the levels of PTHrP were associated with poor prognosis independently of overt hypercalcemia. Hence, one could assume that PTHrP interferes with calcium levels within the normal range [37]. In accordance, the antibody neutralization of PTHrP in mice bearing tumors improved survival [38]. In another animal model, PTHrP showed great influence on tumorigenesis, progression, and metastasis formation in breast cancer xenografts [39]. Moreover, Carriere et al. [40] recently suggested the involvement of PTHrP, secreted protein acidic and rich in cysteine (SPARC), and epithelial–mesenchymal transition (EMT) in CRC, favoring a more aggressive phenotype of the disease. Consistent with the idea of an unmediated effect of calcium levels on survival outcomes, our assessment of inflammatory indexes revealed the opposite results. While patients with stage IV or recurrent CRC with higher calcium levels had greater inflammation, in patients with local and locoregional disease, higher calcium levels were associated with lower inflammatory rates. This suggests that calcium levels do not modulate the inflammatory milieu of the host; rather, the higher calcium levels observed in metastatic patients may be a consequence of the greater tumor burden.

The strength of our study is that it is the first to separately report this association in patients with both locoregional and advanced CRC. Nonetheless, our study has limitations. First, the retrospective nature of this study impeded any further analysis of the mechanisms involved in the association of corrected calcium with prognosis, such as the measurement of PTHrP. Second, the generalizability of our study is limited. We conducted our study in a single institution, and patients attended HC-UNICAMP; these patients represent a population in São Paulo that does not have private insurance and thus does not represent the higher socioeconomic spectra. Third, although the calcium and albumin measurements were obtained in a manner that was dependent on the assistant physician's choice, the notable number of missing data points in our cohort (of the 1552 patients in this cohort, only 498 had calcium and albumin measurements at the given time) may have potentiated unrecognized changes in clinical practice during the study timeframe. Finally, our findings need to be tested in future studies that examine other populations, which must include information regarding PTHrP, calcium, PTH, and vitamin D levels.

In summary, we demonstrated that higher corrected calcium levels might be associated with worse CRC survival outcomes. Although reverse causality may have contributed to our findings, the use of corrected calcium levels as a biomarker of CRC prognosis holds promise for better understanding the mechanisms of CRC aggressiveness and deserves further evaluation in prospective trials to be implemented as a prognostic predictor in clinical practice.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jcm11102928/s1, Table S1: Selected demographic and disease characteristics according to calcium levels of stage I–III colorectal cancer patients; Table S2: Body composition and inflammatory indexes according to calcium tertiles of metastatic colorectal cancer patients; Table S3: Body composition and inflammatory indexes according to calcium tertiles of stage I–III colorectal cancer patients

**Author Contributions:** Conceptualization, M.N.S., M.C.S.M., L.P.d.C. and J.B.C.C.; methodology, M.N.S., L.P., M.C.S.M., L.P.d.C., L.T.M. and J.B.C.C.; software, M.N.S., L.P., M.C.S.M., L.P.d.C. and J.B.C.C.; validation, M.N.S., L.P., M.C.S.M., L.P.d.C. and J.B.C.C.; formal analysis, M.N.S., M.C.S.M. and J.B.C.C.; investigation, M.N.S., L.P., M.C.S.M., L.P.d.C. and J.B.C.C.; resources, M.N.S., L.P., M.C.S.M., L.P.d.C. and J.B.C.C.; data curation, M.N.S., L.P., M.C.S.M., L.T.M. and J.B.C.C.; writing original draft preparation, M.N.S., M.C.S.M.,L.P.d.C. and J.B.C.C.; writing—review and editing, M.N.S., L.P., M.C.S.M., L.P.d.C., F.O.C., L.T.M., S.R.B. and J.B.C.C.; visualization, M.N.S., M.C.S.M. and J.B.C.C.; supervision, M.N.S., M.C.S.M. and J.B.C.C.; project administration, M.N.S., M.C.S.M. and J.B.C.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** The MNS was funded by Conselho nacional de desenvolvimento científico e tecnológico (CNPq), grant number 140596/2019-4; The JBCC was funded by CNPq, grant number 303429/2021-6, and funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant number 2018/23428-0.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of State University of Campinas (CAAE number: 15505419.1.0000.5404, approved in 5 July 2019).

**Informed Consent Statement:** Patient consent was waived due to this is a retrospective work and the research method used guaranteed the anonymity of the participants, as the search was carried out using the identification number generated in HC-UNICAMP for the records hospital. The collected data were stored in a restricted location, and accessed only by the work researchers.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **The Significance of Selected C-C Motif Chemokine Ligands in Colorectal Cancer Patients**

**Monika Zajkowska 1,\*, Maciej Dulewicz 1, Agnieszka Kulczy ´nska-Przybik 1, Kamil Safiejko 2, Marcin Juchimiuk 2, Marzena Konopko 2, Leszek Kozłowski <sup>2</sup> and Barbara Mroczko 1,3**


**Abstract:** Colorectal cancer (CRC) is one of the most frequently diagnosed neoplasms. Despite the advances in diagnostic tools and treatments, the number of CRC cases is increasing. Therefore, it is vital to search for new parameters that could be useful in its diagnosis. Thus, we wanted to assess the usefulness of selected CC chemokines (CCL2, CCL4, and CCL15) in CRC. The study included 115 subjects (75 CRC patients and 40 healthy volunteers). The serum concentrations of all parameters were measured using a multiplexing method (Luminex). The CRP levels were determined by immunoturbidimetry, and the classical tumor markers (CEA and CA 19-9) were measured using CMIA (chemiluminescent microparticle immunoassay). The concentrations of all parameters were higher in the CRC group when compared to the healthy controls. The diagnostic sensitivity, specificity, positive and negative predictive value, and area under the ROC curve (AUC) of all estimated CC chemokines were higher than those of CA 19-9. Interestingly, the obtained results also suggest CCL2's significance in the determination of local metastases and CCL4's significance in the determination of distant metastases. However, further studies concerning the role of selected CC chemokines in the course of colorectal cancer are necessary to confirm and to fully clarify their diagnostic utility and their clinical application as markers of CRC development.

**Keywords:** CRC; CCL2; CCL4; CCL15; diagnostic utility

#### **1. Introduction**

Colorectal cancer (CRC) is one of the most frequent malignancies worldwide, being the second most common malignancy in men and third in women, and accounting for almost 11% and over 9% of all cancer cases, respectively. According to the World Health Organization (WHO), the global incidence of CRC is almost 2 million new cases per year, with approximately 920,000 deaths annually. Importantly, there is an observed increase in both the incidence and the mortality of colorectal cancer, as estimated year-to-year. It was predicted by WHO that the number of new CRC cases may exceed 3,000,000 in 2040, with the number of fatalities reaching 1.5 million per year [1,2]. What should be stressed is CRC is a preventable disease—even in up to 50% of cases—by some modifications of lifestyle, such as a high-fiber and a balanced diet, moderate physical activity, or avoidance of alcohol or smoking [3,4].

Diagnosis of colorectal cancer as early as possible, particularly in the asymptomatic stages, when the tumor is still non-malignant, and initiation of appropriate treatment is of principal importance for patients' survival. The currently used methods of CRC detection include colonoscopy and sigmoidoscopy, as well as imaging diagnostics with the use of

**Citation:** Zajkowska, M.; Dulewicz, M.; Kulczy ´nska-Przybik, A.; Safiejko, K.; Juchimiuk, M.; Konopko, M.; Kozłowski, L.; Mroczko, B. The Significance of Selected C-C Motif Chemokine Ligands in Colorectal Cancer Patients. *J. Clin. Med.* **2022**, *11*, 1794. https://doi.org/10.3390/ jcm11071794

Academic Editors: Ugo Grossi and Milo Frattini

Received: 8 March 2022 Accepted: 23 March 2022 Published: 24 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

computed tomographic colonography and the magnetic resonance method. Although substantial progress has been made in this field in recent years, in the case of small lesions, with a mass not exceeding 1 g, these techniques may be ineffective. Another diagnostic tool useful in the detection of colorectal cancer are tumor markers, mainly glycoproteins or enzymes, which are synthesized by tumor cells. Tumor markers have a particular utility not only in detecting of malignancies and determining tumor advancement, but also in monitoring of treatment and early detection of recurrence [5,6]. The example of tumor markers employed in the diagnosis of CRC are CEA and CA 19-9. Unfortunately, the diagnostic usefulness of these biomarkers is relatively low and they are not specific to the colorectal cancer only [7]. Taking into account the above premises, there is an urgent need to find new diagnostic markers, the use of which will allow for detection of a developing cancer even earlier than before.

Increasing evidence suggests that small (8–12 kDa) inflammatory proteins known as chemokines are key regulators of angiogenesis, including pathological angiogenesis. They are a large family composed of 50 members. The main role of these cytokines is to direct the recruitment and the relocation of cells to locations of inflammation or injury. They are divided into four classes according to the location and the number of cysteine residues at the amino terminus. The CC-chemokine group has two adjacent cysteine residues, and the CXC chemokine group has two cysteine residues detached by an amino acid. The chemokine CX3C group has 3 amino acids between 2 cysteine residues and the C-chemokine group has only1 cysteine residue at the amino terminus. Of the four chemokine groups, the largest group is the CC chemokine group which includes a total of 28 members across all species. This is followed by the chemokine CXC group (17 members), with chemokines CX3C and XC having 1 and 2 members, respectively. All of these proteins exert their biological properties by interacting with G-protein-coupled transmembrane chemokine receptors found on the cell membrane of specific effector cells. The nomenclature of chemokines and their receptors results directly from their classification. At present, there are 19 receptors corresponding to specific groups of chemokines. Despite their large number, these receptors are structurally similar and they are activated in an analogous manner to the chemokines themselves [8].

The current state of knowledge allows us to suspect that chemokines and their receptors play a significant role in cancer development [9]. In tumor growth and metastasis, chemokines and their receptors exert a multifaceted effect on regulating angiogenesis, tumor cell proliferation, and apoptosis, mediating tumor cell metastasis in an organ-specific manner [10]. It is postulated that the CC and CXC chemokines could be the most active in the regulation of angiogenesis [11,12]. That is why the aim of our study was an attempt to clarify and to assess the usefulness of selected CC-chemokine measurement (CCL2, CCL4 and CCL15) in patients with colorectal cancer compared to the healthy volunteer group. We have also compared the obtained results to comparative, routinely used tumor markers (CA 19-9, CEA) and CRP (C-reactive protein), which is an inflammatory parameter.

#### **2. Materials and Methods**

#### *2.1. Patients*

The study included 75 colorectal cancer (CRC) patients diagnosed by the oncology group (Table 1). The patients were treated in the Department of Oncological Surgery with Specialized Cancer Treatment Units, Maria Sklodowska-Curie Oncology Center, Bialystok, Poland. Tumor classification and staging were conducted in agreement with the International Union Against Cancer Tumor–Node–Metastasis (UICC-TNM) classification. The histopathology of colorectal cancer was based on the examination of tissue samples with the use of a microscope. Moreover, all patients were grouped according to not only tumor stage (TNM), but also depth of tumor invasion (T factor), the presence of lymph node (N factor), and distant metastases (M factor), as well as the histological grade (G factor) of the tumor. The pretreatment staging procedures included physical and blood examinations, computed tomography (CT) and—in case of patients with rectal cancer—magnetic resonance imaging

(MRI) of the small pelvis. Additionally, all patients were assessed according to the Eastern Cooperative Oncology Group (ECOG) score. The control group comprised 40 healthy volunteers. For each patient qualified for the control group, the following exclusion criteria was applied: active infections and symptoms of an infection (both bacterial and viral); other comorbidities which can affect cytokine concentrations (respiratory diseases, digestive tract diseases); or systemic diseases such as lupus, rheumatoid arthritis, or collagenosis.


**Table 1.** Characteristics of colorectal cancer and healthy patient groups.

#### *2.2. Biochemical Analyses*

Venous blood samples were collected from each patient into a tube with a clot activator (S-Monovette, SARSTEDT, Numbrecht, Germany), centrifuged to obtain serum samples, and stored at −80 ◦C until assayed. The tested chemokines were measured with the use of a Luminex 200 analyzer (Thermo Fisher Scientific, Waltham, MA, USA) (multiplexing, multiparametric, fluorescence laser reading system on microspheres for the simultaneous determination of multiple parameters) and Luminex Human Discovery assay plates, provided by R&D systems, Abingdon, UK. According to the manufacturer's protocols, duplicate samples were assessed for each standard, control, and sample. The serum levels of classical tumor markers were measured with chemiluminescent microparticle immunoassay (CMIA) (Abbott, Chicago, IL, USA); and, for the analysis of the CRP concentration, the immunoturbidymetric method (Abbott, Chicago, IL, USA) was used according to the manufacturer's protocols.

#### *2.3. Statistical Analysis*

Statistical analysis was performed by RStudio. The introductory statistical analysis (using the Shapiro–Wilk test) exposed that the tested parameters and tumor marker levels did not follow normal distribution. Therefore, statistical analysis between the groups was performed with the use of the U Mann–Whitney test, the Kruskal–Wallis test, and a multivariate analysis of various data by the post-hoc Dwass–Steele–Critchlow–Flinger test. The data were presented as a median and a range. Diagnostic sensitivity, specificity, and the predictive values of positive and negative test results (SE, SP, PPV, and NPV, respectively) were calculated by using the cut-off values which were calculated by the Youden's index (as a criterion for selecting the optimum cut-off point) and for each of the tested parameters they were as follows: CCL2—426.13 pg/mL, CCL4—274.45 pg/mL, CCL15—2607.49 pg/mL, CA 19-9—5.30 U/mL, CEA—1.70 ng/mL, and CRP—2.50 mg/L. We also defined the receiver operating characteristics (ROC) curve for all of the tested parameters, tumor markers, and for the CRP to estimate diagnostic accuracy, and we performed a Spearman's rank correlation test. Statistically significant differences were defined as comparisons resulting in *p* < 0.05.

#### **3. Results**

Table 2 shows the serum levels of the CCL2, CCL4, CCL15, CA 19-9, CEA, and CRP in patients with colorectal cancer and in the control group. After performing the nonparametric U Mann–Whitney test comparing the concentrations obtained in both groups, we observed that the levels of CCL2, CCL4, CEA, and CRP in the entire cancer group were significantly higher (in all cases *p* < 0.05).


**Table 2.** Serum levels of tested parameters in cancer and control groups.

\* U Mann–Whitney test; CCL—chemoattractant cytokine ligand; CA 19-9—cancer antigen 19-9; CEA carcinoembryonic antigen; CRP—C-reactive protein. The statistically significant results are presented in bold.

In addition, we performed a more thorough analysis with use of Kruskal–Wallis and Dwass–Steel–Critchlow–Fligner tests after the division of the total CRC group into advancement groups (TNM I-IV). As a result of this analysis, we obtained significant results for almost all parameters (Table 3). Interpreting the obtained results, it can be suggested that the concentration of CCL4, CEA, and CA 19-9 increases significantly with the advancement of neoplastic changes, and it may be related not only to the number of neoplastic cells, but also to their spread—as TNM stage III is associated with the presence of metastases to nearby lymph nodes and stage IV with distant metastases. Interestingly, the CRP analysis confirms the inflammatory theory of neoplasm, as statistically significant differences were

obtained only in the case of comparisons between the control group and individual stages of cancer.


**Table 3.** Kruskal–Wallis and Dwass–Steel–Critchlow–Fligner tests analysis results.

The statistically significant results are presented in bold.

Considering the fact that in the subgroups of TNM stages I and II, the number of patients did not exceed 20, which may affect the accuracy of the obtained results, we decided to confirm them using the U Mann–Whitney test. We divided the group of all CRC patients into the group of less-advanced neoplasms (TNM I + II) and the group of advanced neoplasms (TNM III + IV). In addition, we divided the group of advanced neoplasms into separate TNM stages (III and IV) due to the sufficient number of patients in each subgroup to perform a precise analysis in different subgroups and in comparison to the control group. The results obtained were similar to those in previous analyses. Interestingly, we observed significant differences between controls and III stage TNM in the case of CCL2, which may suggest its participation in local lymph node metastasis processes. In the case of CCL4, we observed differences between the control and the most advanced stage of CRC, and what is of utmost importance, significant differences between all advancement CRC stages (similarly to CA 19-9). In comparison between the control group and all advancement stages, CEA and CRP revealed significance; but, in case of differences between TNM stages, significant results were obtained only in case of CEA between less-advanced stages and distant stage metastases (Table 4).



The statistically significant results are presented in bold.

Table 5 shows the sensitivity, specificity, positive and negative predictive values (SE; SP; PPV; NPV, respectively), and the relationship between them with the use of the area under the ROC curve (AUC) of all newly tested parameters. We indicated that the highest SE from all parameters revealed CCL4 (76%). The observed value is higher than SE of

commonly used tumor markers such as CEA (75%), CA 19-9 (51%) and C-reactive protein (73%). In the case of SP, the highest value was observed for CCL2 (60%) and it was higher than SP of CA 19-9 (48%), but the highest specificity from all parameters was observed in case of CRP (78%) and CEA (70%). Positive and negative predictive values were highest in case of CCL2 and CCL4 (72%/47% and 74%/53%, respectively). These values were slightly lower than PPV and NPV of CEA and CRP. What is more, the SE, SP, PPV, and NPV values of all newly tested parameters (CCL2, CCL4, CCL15) were higher than CA 19-9, which confirms their higher usefulness in case of patients with CRC than the routinely used marker.


**Table 5.** Diagnostic criteria of tested parameters in patients with colorectal cancer.

SE—sensitivity; SP—specificity; PPV—positive predictive value; NPV—negative predictive value.

We noticed that the CCL2 and CCL4 areas under the ROC curve (0.634; 0.630,respectively) in the entire group of colorectal cancer were highest from all newly tested parameters, but lower than AUC for CEA and CRP. Additionally, similar to previously mentioned statistical parameters, in the case of all tested CC chemokines, AUC was higher than AUC for CA 19-9. A graphical version of all of the significant ROC analysis results is shown in Figure 1. The AUCs for the tested parameters, as for generally used tumor markers and combined analysis, were significantly larger in comparison to AUC = 0.5 (borderline of the diagnostic usefulness of the test) (*p* < 0.05 in all cases).

In order to complete the statistical analysis, we checked the Spearman's rank correlation coefficient to measure and to show the strength and the direction of monotonic association between variables in the CRC group. Obtained results are shown in Table 6. We observed a strong positive correlation for one of the tested parameters (CCL4) and

the tumor TNM stage. This may confirm that the increasing concentration of this parameter is related to the number of neoplastic cells. This fact was also observed during the Kruskal–Wallis and Dwass–Steel–Critchlow–Fligner tests. In the case of the remaining parameters (CEA and CA 19-9), we also observed a similar correlation but of moderate strength. Moderate, positive correlation was also observed between the CEA and the CCL4 concentrations, and concentrations of both markers (CEA and CA 19-9). The rest of the observed correlations revealed weak strength (coefficient < 0.40). Interestingly, we observed also one negative but weak correlation between the CCL2 and the CCL15 concentrations.

**Figure 1.** Receiver operating characteristics for all significant ROC analysis results.



The statistically significant results are presented in bold.

#### **4. Discussion**

At present, in case of patients with colorectal cancer it is clinically important to search for new prognostic or predictive markers, as they might influence postoperative decisions. Generally, the guidelines for CRC are mainly based on the basis of, e.g., the TNM stage or the molecular characteristics of the tumor. In some cases, the decision whether to use or not to use adjuvant chemotherapy requires additional tests such as for a serum CEA level or an expression of p53/Ki67 [13]. In accordance with that, researchers are searching for different, new parameters to find markers for the highly accurate and non-invasive tests for colorectal cancer.

We indicated that the serum concentration for CCL2 was statistically higher in the group of colorectal cancer patients when compared to healthy controls (*p* = 0.02). Similar results were obtained in the work of De la Fuente López et al. [14]. These authors revealed that not only plasma levels, but also the concentration of this parameter in CRC tissue lysates is significantly higher when compared to healthy mucosa. Nevertheless, as the number of samples in these investigations were low (25 tissues; 32 CRC patients and 15 healthy patient plasma samples), these results needed a verification. Another work confirmed that a higher expression of CCL2 can be found in cancer tissue, and it is connected with a negative prognosis in CRC patients [15]. Interestingly, some researchers have indicated that an overexpression of CCL2 is associated with increased metastatic potential [16]. A different study by Nardelli et al. [17] also established that circulating CCL2 levels were associated with the presence of CRC, but the number of patients in this study was also insufficient (20 CRC patients and 20 healthy volunteers). A different research group also suggested that higher CCL2 levels may be considered as a prognostic factor in CRC, but this study was performed with the use of serum from 45 patients, and peculiarly, the control group was not included [13]. Therefore, our research carried out on a much larger group of patients with the use of a sufficiently large group of healthy volunteers, finally confirms the previously mentioned fragmentary reports.

Some studies also assessed the changes of CCL2 concentration after surgery or perioperatively. Hua et al. [18] discovered that elevated levels of this parameter are associated with a high risk of overall mortality. On the contrary, a study by Watanabe et al. [19] revealed that a decrease in the CCL2 ratio between tumoral and normal adjacent tissues is associated with lymph node involvement, and it could predict a poor prognosis. This discrepancy may be related not only to the difference in the material used for research, but also to the calculated ratio. Its reduction may be caused not only by lower expression in neoplastic tissues, but also by increased expression in healthy tissues. Interestingly, when analyzing the concentration of CCL2 in the control group and the study group by TNM stage, we showed a significant relationship between stage III and the control group, which may be a confirmation of the Watanabe et al. [19] investigation, which pointed out the relationship between CCL2 and the appearance of local lymph node metastases. In addition, the work of Johdi et al. [20] showed that there were no differences in the serum concentration of CCL2 and CRC, polyp, and healthy subjects. However, these results were performed on a basis of only 20 samples from each group. In the work of Tonouchi et al. [21], CCL2 levels were significantly raised 1 h after surgery, which suggests that this parameter can have a different role than as a marker of surgical insults, especially, as these differences did not correlate with IL-6 changes. However, a few days after surgery, the levels of this parameter were comparable to those before surgery. Due to discrepancies in the previously obtained studies, all previously mentioned results require further confirmation, which indicates a further plan for the continuation of our research.

We also found studies concerning the CCL2 concentration and expression in murine models [22,23]. These authors concluded that this cytokine may activate macrophages to become tumoricidal, resulting in the suppression of metastasis; and, they could be useful as biomarkers of colon cancer progression, which fully coincides with our discoveries.

On the other hand, one of the publications indicated that CCL2 did not show any differences between the adenoma group compared to the control group. This inconsistency may be related to a too-early period of changes leading to cancer progression. However, the serum samples used in the study were stored for many years and they were transported several times, which may have a significant impact on the results obtained by those researchers [24]. These results also suggest the need for further confirmation.

In the work of De la Fuente López et al. [14], CCL4 concentration was also found (similar to our study) to be significantly increased in CRC patients when compared to healthy controls. The previously mentioned work by Johdi et al. [20] also included the CCL4 determinations. Interestingly, in case of this parameter, significantly higher concentrations in the blood serum of patients with CRC compared to the control group were observed, as well as in the serum of patients with colorectal polyps.

Remarkably, in the work of Krzystek-Korpacka et al. [25], it was shown that CCL4 concentrations in the case of CRC are significantly higher when compared to the control group. However, after division by tumor location (rectum and colon), it turned out that in the case of rectal cancer, these concentrations are the highest and the difference is statistically significant. This can be important information when attempting to personalize therapy, and it is indicative of the heterogeneity of CRC. Surprisingly, in the work of Pervaiz et al. [26], completely contradictory results were presented. In IV stage of the tumor's advancement, statistically lower concentrations of CCL4 were demonstrated compared to the control group. These differences may be related to the number of patients, as the studies of Pervaiz et al. [26] were carried out on a group of 24 patients diagnosed with CRC, of which only 3 were at stage IV of CRC advancement.

In the case of CCL15, for which we did not observe any statistical differences, we found only one study that assessed the concentration of this parameter in the course of CRC. Inamoto et al. [27] showed that the concentrations of CCL15 in patients with CRC are statistically significantly higher than in healthy subjects, not only in the entire study group, but also at various stages of CRC advancement. These differences are extremely difficult to explain, as both experiments involved a properly large group of patients. However, the results obtained by Inamoto et al. [27] were several times higher (median for the control group 9.4 ng/mL; for the tested group 17.8 ng/mL). Possibly these differences could be influenced by the method of determination (ELISA vs. Luminex) or the ethnicity of the patients (Asian vs. European).

Unfortunately, we have not found any other papers that would focus on demonstrating the dependence and statistical significance based on the division of the study group into stages of advancement. Therefore, we believe that our work is innovative in this matter, which significantly increases its value. A more accurate demonstration of the relationships between the control group and the study group may significantly affect the understanding of changes in the course of CRC. Interestingly, our results showed a significant relationship between CCL2 and III TNM stage of CRC, which may be associated with the formation of local lymph nodes metastases, and significant differences between the concentration of CCL4 in stage IV of CRC and the control group, which may indicate its involvement in the development of distant metastasis. Due to the fact that these are the first reports on these dependencies, it is advisable to confirm them in further analyses.

According to our knowledge, the present study is also the first that assesses diagnostic criteria such as SE, SP, PPV, NPV, and ROC. However, parameters such as CCL2 and CCL4 showed high values (especially diagnostic sensitivity) compared to markers routinely used in diagnostics, and even higher than CA 19-9. This is significant evidence that these cytokines can contribute to the development of diagnostics and constitute an additional diagnostic parameter, e.g., in the case of detecting local and distant metastases. Perhaps a simultaneous analysis of the classical tumor markers and the tested cytokines would increase their diagnostic utility, which is an important task for the continuation of our research in the future. The only work assessing merely the SE and the SP of the CCL15 chemokine was the previously mentioned work of Inamoto et al. [27], whose results were significantly higher than ours (78.8%; 70% vs. 57%; 53%, respectively). These discrepancies may be due to the same reasons as for the concentrations of CCL15 described above.

We also tried to determine the correlations between the examined parameters, which showed that the concentration of CCL2 positively correlates with the concentration of CCL4 and negatively correlates with the concentration of CCL15. In contrast, CCL4 positively correlated with routine markers (CEA, CA 19-9), CRP protein, age (similar to CEA), and tumor stage (similar to CEA and CA 19-9). The study by De la Fuente López et al. [14] showed a significant correlation between CCL4 and the CD163 marker on pro-tumor macrophages and inflammatory mediators (VEGF, TNF-α). This indicates the high potential of CCL4 to induce infiltration of tumor-associated macrophages which may be related to tumor progression or metastases associated with high levels of CCL4, which was found in our study.

#### **5. Conclusions**

According to our knowledge, the current study is the first that links the diagnostic characteristics of CCL2, CCL4, and CCL15 with the well-established, classical tumor markers (CEA and CA 19-9) and CRP—which is the marker of inflammation—in CRC patients, and not only in the entire study group, but also in subjects divided according to TNM stage. The results obtained suggest the significant importance of CCL2 in the determination of local metastases and CCL4 in the case of distant metastases. However, after a careful analysis of our results and the results of other authors, it is certain that further studies concerning the concentrations of selected CC chemokines in the course of colorectal cancer are necessary to confirm and to clarify their diagnostic utility and their clinical application as potential non-invasive markers of CRC development.

**Author Contributions:** Conceptualization, M.Z. and B.M.; methodology, M.Z. and A.K.-P.; formal analysis, M.Z., A.K.-P. and M.D.; investigation, M.Z., M.D., K.S., M.J., L.K., M.K. and A.K.-P.; resources, M.Z.; data curation, M.Z. and M.D.; writing—original draft preparation, M.Z.; writing—review and editing, M.Z. and B.M.; supervision, B.M.; project administration, M.Z. and B.M.; funding acquisition, M.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Medical University of Bialystok, Poland (grant numbers: SUB/1/DN/21/004/1198 and SUB/1/DN/22/004/1198).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and it was approved by the Ethics Committee of the Medical University of Bialystok (R-I-002/564/2019; 28 November 2019).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. Key data are stated in the text.

**Acknowledgments:** This study was conducted with the use of equipment purchased by the Medical University of Bialystok as part of RPOWP 2007-2013 funding, Priority I, Axis 1.1, contract No. UDA-RPPD.01.01.00-20-001/15-00 dated 26.06.2015. B.M. received consultation and/or lecture honoraria from Abbott, Wiener, Roche, Cormay, and Biameditek. M.Z. received lecture honoraria from Roche.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Yu-Min Huang 1,2, Po-Li Wei 1,3, Chung-Han Ho 4,5,† and Chih-Ching Yeh 6,7,8,9,\*,†**


**Abstract:** We investigate whether cigarette smoking is associated with survival in patients with colorectal cancer (CRC) through a nationwide population-based cohort study in Taiwan. The Taiwan Cancer Registry and National Health Insurance Research Database were used to identify data from patients with CRC from 2011 to 2017. Tobacco use was evaluated based on the smoking status, intensity, and duration before cancer diagnosis. A total of 18,816 patients was included. A Kaplan– Meier survival analysis indicated smoking to be significantly associated with the CRC mortality risk (log-rank *p* = 0.0001). A multivariable Cox model indicated that smoking patients had a 1.11-fold higher mortality risk (HR = 1.11, 95% CI = 1.05–1.19) than nonsmoking patients did. This increased risk was also present in patients with CRC who smoked 11–20 cigarettes per day (HR = 1.16; 95% CI = 1.07–1.26) or smoked for >30 years (HR = 1.14; 95% CI = 1.04–1.25). Stratified analyses of sex and cancer subsites indicated that the effects of smoking were higher in male patients and in those with colon cancer. Our results indicate that cigarette smoking is significantly associated with poor survival in patients with CRC. An integrated smoking cessation campaign is warranted to prevent CRC mortality.

**Keywords:** colorectal cancer; cigarette smoking; survival

#### **1. Introduction**

Colorectal cancer (CRC) is among the most common cancers and among the leading causes of cancer deaths worldwide [1–4]. Although it has historically been more prevalent in the West, the incidence rates of CRC have been increasing in East Asian countries [5]. In Taiwan, CRC is one of the most commonly diagnosed cancers [6]. Despite the progress that has been achieved in its diagnosis and treatment, approximately half of patients with CRC die within 5 years of diagnosis [4]. Therefore, further efforts to identify and obviate the risk factors of CRC mortality are required to improve the prognosis of this cancer.

Cigarette smoking is a serious public health concern; it is annually responsible for millions of deaths around the world [7]. Smoking is estimated to be responsible for more than 30% of cancer deaths in the United States each year. Smoking has also been observed to increase the risk of mortality in CRC [8]. The association between smoking and CRC

**Citation:** Huang, Y.-M.; Wei, P.-L.; Ho, C.-H.; Yeh, C.-C. Cigarette Smoking Associated with Colorectal Cancer Survival: A Nationwide, Population-Based Cohort Study. *J. Clin. Med.* **2022**, *11*, 913. https:// doi.org/10.3390/jcm11040913

Academic Editor: Ugo Grossi

Received: 11 December 2021 Accepted: 7 February 2022 Published: 9 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

has been demonstrated in many studies [9–13]. Long-term smokers have been reported to have a significantly increased risk of developing CRC than nonsmokers [13–16]. Studies have reported a 15% to 60% higher risk estimate associated with active smoking [1,17,18]. Although data were insufficient for the association between smoking and CRC to be defined as casual, recent studies have suggested cigarette smoking to be a risk factor for CRC [1,4,13,19–21]. Consequently, the American College of Gastroenterology colorectal cancer screening guidelines have highlighted smokers as being at an increased risk [22].

Cigarette smoking may worsen the prognosis of CRC [23,24]. Long-term cigarette smoking has been reported to increase the risk of both overall and disease-specific CRC mortality in men and women [4,9,13,19]. However, findings regarding the influence of smoking on CRC survival have been inconsistent; several studies have also reported no significant association between smoking and CRC mortality [25,26].

Moreover, many of the aforementioned studies on the association between smoking and the risk and prognosis of CRC were conducted in Western countries [5,27]. In the 16 studies included in one meta-analysis, only 1 was conducted in East Asia [28]. Therefore, because of factors such as ethnicity, culture, and lifestyle, the reported findings of this meta-analysis may not be directly applicable to other demographic groups. Another meta-analysis reported that the relative risks (RRs) of CRC among current smokers were significantly different in different geographic areas [1]. In addition, the results of studies from Asian countries have generally been heterogeneous, which further complicates the matter [5]. Evidence regarding the effects of cigarette smoking on the prognosis of CRC remains limited. Therefore, we perform a nationwide population-based cohort study to investigate whether cigarette smoking adversely affects the survival outcomes of Asian patients with CRC.

#### **2. Materials and Methods**

#### *2.1. Data Sources*

Data in this study were collected from the Taiwan Cancer Registry (TCR) and Taiwan's National Health Insurance Research Database (NHIRD). Both of these databases are managed by the Health and Welfare Data Science Center (HWDC) of the Ministry of Health and Welfare. The TCR was established to gather information on individual demographics, cancer stages (AJCC 7th edition), primary sites, histology, and treatment types in patients with cancer to understand the incidence and mortality rates of cancer in Taiwan. The NHIRD was established for research purposes; it contains data from Taiwan's single-payer insurance system, in which more than 99% of Taiwan's 23 million citizens are registered. The NHIRD contains registration files and original inpatient and outpatient reimbursement claim data from 1996 to 2017. The datasets of the HWDC are all de-identified forms. This study was conducted in compliance with the Declaration of Helsinki of 1964 and was approved by the Research Ethics Committee of Chi Mei Hospital (IRB no. 10702-E04). The requirement for informed consent was waived by the Research Ethics Committee of Chi Mei Hospital.

#### *2.2. Study Population*

The TCR was used to identify patients with CRC based on the International Classification of Diseases for Oncology, third edition (ICD-O-3); in this study, colon (ICD-O-3: C18), rectosigmoid junction (ICD-O-3: C19), and rectum (ICD-O-3: C20) cancers were included. Because the TCR began recording smoking and drinking behavioral information in 2011, patient data from 2011 to 2017 were selected. Patients with a history of CRC before 2011 were excluded to reduce omitted variable bias. In addition, because the aim of this study was to estimate the association between cigarette smoking and risk of mortality in patients with CRC, included patients were categorized as those with and without a history of prediagnostic smoking. Those with a smoking history included both current and ever smokers, for whom the duration of smoking in years and smoking count per day were included in the analysis. To reduce the potential confounding factors of mortality, including

age, gender, clinical stage, grade, and cancer subsite, between patients with smoking and those without, we randomly selected two patients without smoking to match each patient with smoking using propensity score approach. A propensity score matching approach with the nearest-neighbor matching algorithm was used in this study according to SAS macro "*%OneToManyMTCH*". The flowchart of the study population selection is presented in Figure 1.

**Figure 1.** Flowchart of study population selection.

#### *2.3. Measurements*

The major outcome of this study was overall mortality. Mortality was defined using Taiwan's cause-of-death data. All patients were right censored to date of death or 31 December 2017, whichever came first. The study variables, namely, age at diagnosis, sex, clinical stage, histological grade, and alcohol drinking habit, were all collected from the TCR. Age was divided into groups of <40, 40–49, 50–59, 60–69, and ≥70 years. Charlson comorbidity index (CCI) scores were calculated using patients' diagnosis records from the NHIRD to represent patients' comorbidities, which were defined before the date of diagnosis of CRC. To generate the index score, each of the 19 identified medical conditions was scored from 1 to 6.

#### *2.4. Statistical Analysis*

The frequency was presented as a percentage for categorical variables among the study population. The distribution difference between smoking and nonsmoking groups was compared using Pearson's chi-square test. The trend of mortality during the study period was plotted using the Kaplan–Meier approach, with a log-rank test for estimating the statistical difference between smoking and nonsmoking patients with CRC. Multivariable Cox proportional regression was constructed to estimate the mortality risk and control for potential confounders by adjusting for age, sex, drinking habit, residence in a remote area, cancer site, cancer clinical stage, cancer grade, and CCI group. Stratified analyses of age, sex, and CRC subsites were also presented. To observe the progress of mortality risk on smoking counts per day and smoking years, the linear trend test was used to estimate the potential trends. All analyses were conducted using SAS statistical software version 9.4 (SAS Institute, Cary, NC, USA). Significance was set at *p* < 0.05. Kaplan–Meier curves were plotted using STATA (version 12; Stata, College Station, TX, USA).

#### **3. Results**

#### *3.1. Characteristics of Study Population*

The baseline characteristics of the matched cohort are presented in Table 1. Of the 18,816 patients with CRC included in this study, 6272 were smokers and 12,544 were not. The smoking group comprised more patients with drinking habits (52.1% vs. 13.8%, *p* < 0.0001). In addition, the mortality rate was significantly higher in the smoking group (30.1% vs. 27.9%, *p* = 0.0012). Otherwise, the two groups were balanced with regard to age, sex, residence in remote area, cancer subsite, clinical stage, tumor grade, and CCI grouping.

**Table 1.** Demographic analysis of smoking and nonsmoking patients with colorectal cancer.


#### *3.2. Cigarette Smoking and Mortality Risk*

As illustrated in Figure 2, a significant difference was found in the mortality risk of CRC between the smoking and nonsmoking groups (log-rank test *p* = 0.0001). The crude data, presented in Table 2, revealed that smoking patients had a 1.11-fold higher mortality risk (95% CI = 1.05–1.19; *p* = 0.0009) than nonsmoking patients did. Regarding the effects of smoking intensity, patients who smoked 11–20 cigarettes per day (HR = 1.17; 95% CI = 1.08–1.27; *p* = 0.0001) and who smoked for more than 10 years (HR = 1.12; 95% CI = 1.02–1.23; *p* = 0.0184 for patients smoking for 11–30 years; HR = 1.15; 95% CI = 1.06–1.26; *p* = 0.0014 for those smoking for >30 years) had a significantly higher mortality risk than nonsmoking patients did. After adjustment for age, sex, alcohol-drinking habit, residence in remote areas, cancer subsites, cancer clinical stage, cancer tumor grade, and CCI score grouping, smoking patients had a 1.10-fold higher mortality risk (95% CI = 1.03–1.18; *p* = 0.0056) than nonsmoking patients did. Patients who smoked 11–20 cigarettes (HR = 1.16; 95% CI = 1.07–1.26; *p* = 0.0006) per day and who smoked for more than 10 years (HR = 1.11; 95% CI = 1.01–1.23; *p* = 0.0356 for patients smoking for 11–30 years; HR = 1.14; 95% CI = 1.04–1.25; *p* = 0.0044 for those smoking for >30 years) had a significantly higher mortality risk than nonsmoking patients did. A significant trend was identified for increased mortality risk due to smoking duration (*p* = 0.0474).

**Figure 2.** Association of cigarette smoking with mortality risk of colorectal cancer.

**Table 2.** Cigarette smoking associated with mortality risk of colorectal cancer.


**<sup>a</sup>** Adjusted for age (continuous), sex, drinking habit, residence in remote areas, cancer subsite, cancer clinical stage, cancer grade, and CCI group.

#### *3.3. Cigarette Smoking and Mortality Risk Stratified by Sex*

The data presented in Table 3 revealed the risk of mortality associated with smoking in patients with CRC stratified by sex. The proportions of male and female smokers were the same (33.3%), but the male smoking population was 9.47 times of the female. Smoking men had a 1.09-fold higher mortality risk (95% CI = 1.02–1.18; *p* = 0.0156) than nonsmoking men did. A further analysis revealed a significantly higher risk of mortality in men who smoked 11–20 cigarettes per day (HR = 1.16; 95% CI = 1.06–1.26; *p* = 0.0011) and who smoked for more than 10 years (HR = 1.11; 95% CI = 1.01–1.24; *p* = 0.0390 for men smoking for 11–30 years; HR = 1.14; 95% CI = 1.04–1.25; *p* = 0.0076 for those smoking for >30 years) than in those who were nonsmokers. For women, a 1.59-fold higher mortality risk was observed only in those who smoked for 1–10 years (95% CI = 1.03–2.45; *p* = 0.0367) compared with nonsmoking women. However, the increase in risk was not significant in the other levels of smoking intensity.

**Table 3.** Cigarette smoking associated with mortality risk of colorectal cancer stratified by sex.


**<sup>a</sup>** Adjusted for age (continuous), drinking habit, residence in remote areas, cancer subsite, cancer clinical stage, cancer grade, and CCI group.

#### *3.4. Cigarette Smoking and Mortality Risk Stratified by Cancer Subsite*

The risk of mortality associated with smoking with respect to the CRC subsite is presented in Table 4. Smoking patients were associated with a significantly higher mortality risk than nonsmoking patients were for colon (HR = 1.12; 95% CI = 1.03–1.22; *p* = 0.0096) but not rectal cancers (HR = 1.08; 95% CI = 0.95–1.22; *p* = 0.2339). Furthermore, patients with colon cancer who smoked more than 10 cigarettes (HR = 1.15; 95% CI = 1.04–1.28; *p* = 0.0072 for patients smoking 11–20 cigarettes; HR = 1.18; 95% CI = 1.01–1.39; *p* = 0.0376 for those smoking >20 cigarettes) daily and who smoked for more than 10 years (HR = 1.13; 95% CI = 1.00–1.28; *p* = 0.0447 for patients smoking for 11–30 years; HR = 1.18; 95% CI = 1.05–1.32; *p* = 0.0046 for those smoking for >30 years) had a significantly higher mortality risk than nonsmoking patients did. A significant trend was also identified for an increased mortality risk due to smoking intensity (*p* = 0.0463). By contrast, no significant increase in mortality risk was observed in patients with rectal cancer except in those smoking 11–20 cigarettes per day (HR = 1.17; 95% CI = 1.01–1.35; *p* = 0.0337).


**Table 4.** Cigarette smoking associated with mortality risk of colorectal cancer stratified by cancer subsite.

**<sup>a</sup>** Adjusted for age (continuous), sex, drinking habit, residence in remote areas, cancer clinical stage, cancer grade, and CCI group.

#### *3.5. Cigarette Smoking and Mortality Risk Stratified by Age*

The effects of smoking on the mortality risk were assessed with the stratification of the patients' ages (Table 5). For patients younger than 60, an increased CRC mortality risk was not associated with smoking, with the exception of those who smoked for more than 30 years (HR = 1.43; 95% CI = 1.10–1.87; *p* = 0.0074). By contrast, smoking significantly increased the risk of mortality in patients with CRC who were older than 60 (HR = 1.12; 95% CI = 1.03–1.21; *p* = 0.0061). Higher risks were observed in such patients who smoked 11–20 cigarettes per day (HR = 1.16; 95% CI = 1.05–1.27; *p* = 0.0034) and who smoked for more than 10 years (HR = 1.15; 95% CI = 1.02–1.30; *p* = 0.0190 for patients who smoked for 11–30 years; HR = 1.12; 95% CI = 1.02–1.24; *p* = 0.0209 for those who smoked for more than 30 years).

**Table 5.** Cigarette smoking associated with mortality risk of colorectal cancer stratified by age.



**Table 5.** *Cont.*

**<sup>a</sup>** Adjusted for age (continuous), sex, drinking habit, residence in remote areas, cancer subsite, cancer clinical stage, cancer grade, and CCI group.

#### **4. Discussion**

Through the combined analysis of data from nationwide health insurance and cancer registries, we demonstrated that cigarette smoking was associated with a significantly increased risk of mortality in patients with CRC. The increased risk was more prominent in patients with higher levels of smoking intensity and duration. This pattern was especially present in men, patients with colon cancer, and patients older than 60. A dose–response effect on the risk of mortality was also observed with smoking duration in the whole population and with smoking amount in patients with colon cancer. Although the increase in the mortality risk was moderate in most of the analyzed categories, its significance merits further consideration to improve the prognosis of CRC.

Despite widespread skepticism towards an association between cigarette smoking and CRC, accumulating evidence has suggested an increased risk of incidence incurred with smoking [5]. In the Iowa Women's Health Study (IWHS), ever smokers had a moderately increased CRC risk (RR of approximately 1.20) compared with never smokers [11,27]. In the Cancer Prevention Study II Nutrition Cohort, the incidence of CRC was approximately 30% higher in current smokers than in never smokers [18]. Two meta-analyses demonstrated that the pooled RR increased from 15% to 20% in ever smokers compared with never smokers [1,16].

Furthermore, cigarette smoking was associated with an increased risk of mortality in patients with CRC. In the IWHS, ever smokers had an increased risk of overall mortality (RR = 1.31) compared with never smokers, which was similar to observations of CRC incidence [11]. In a previous meta-analysis, current smokers exhibited a significantly higher risk of CRC mortality (RR = 1.58) compared with nonsmokers [16]. In addition to allcause mortality, disease-specific mortality was affected by current smoking [23,24]. Further evidence of this was presented in a meta-analysis that demonstrated that smokers had a 26% higher risk of all-cause mortality than never smokers did. Notably, 30-day mortality was reported to be higher by between 49% and 100% [4]. Compared with these previous studies, the mortality risk increased only moderately, though significantly, in smokers in our study. Factors such as components in cigarettes or differences in the study population might be implicated.

Our results indicated an increased risk of mortality in patients who smoked more than 10 cigarettes per day and who smoked for more than 10 years. This amount and the duration were much lower than previously reported. In the Chicago Heart Association cohort, a significant association between smoking and an increased CRC mortality was observed only in patients who smoked more than 20 cigarettes per day [19]. Smoking more than 15 cigarettes per day and having a 20-pack-year history were reported by Walter et al. to affect CRC survival [4]. In addition to differences in the characteristics of cigarettes and study populations, the lower threshold of smoking intensity for an increasing CRC

mortality risk in our study may be attributable to the numerous events that may increase the detectability of differences in risks.

The mechanisms underlying the association between smoking and CRC mortality are multifold and incompletely understood. Cigarette smoke contains more than 60 carcinogens [7,17,29]. Of them, nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone may enhance metastasis, which is the leading cause of death in patients with CRC, by enhancing cell migration and epithelial–mesenchymal transformation [6,30,31]. Nicotine may also interfere with the antiproliferative and proapoptotic effects of chemotherapeutic agents [4,32,33]. Tobacco smoking may cause a mutation of the GSTM1 gene, resulting in the impaired detoxification of tobacco carcinogens and enhancement of carcinogens' tumorigenic actions. Smoking may also induce aberrant promoter DNA methylation and silence regulatory genes in tumor progression. Consequent genetic alterations, such as a high microsatellite instability (MSI), the CpG island methylator phenotype, and the BRAF V600E mutation, may impair patient survival [13].

In previous studies, risk factors associated with an increased CRC mortality in smokers included an active smoking status, increased smoking amount or duration, and younger age at initiation [4,9,13,18]. The effects of smoking were also more significant in patients younger than 50 [19,24]. Notably, in our study, smoking patients younger than 60 had a lower risk of mortality. An exception to this was the increased risk in those who smoked for more than 30 years, indicating effects of prolonged smoking duration and younger age at initiation. By contrast, a generally significant association was noted between smoking and an increased risk of mortality in patients older than 60. This contradicted the observations by Colangelo et al. that the association between CRC and smoking mortality was significant only in those younger than 50 [19]. Whether this variance can be explained by other unanalyzed factors, such as genetic alterations, requires further elucidation.

For the effect of sex, Colangelo et al. reported that the risk for CRC mortality was higher for women than for men at the same level of smoking exposure, a phenomenon similar to that observed in patients with lung cancer [19]. However, other studies have reported discordant results. The association between smoking and the risk of CRC mortality was higher in men in a study conducted in Canada [13]; the association was even greater in patients older than 60. Walter et al. and Phipps et al. also reported a greater risk of recurrence or mortality in male smokers [4,24]. In our study, the increased risk in male smokers remained significant and relatively constant at most levels of smoking amount and duration. By contrast, the increases in risk in female smokers were mostly nonsignificant. Although our results may suggest a higher CRC mortality risk in male smokers, the disproportionately low number of female smokers when compared with that of male smokers may attenuate the association in females [4,9,11,18,19,21].

Tumor-related factors associated with an increased risks of recurrence or mortality in smokers include a T3 tumor, one to three affected lymph nodes, nonmetastatic diseases, a mutated KRAS status, and a wild-type BRAF status [4,24]. The effect of MSI remains under debate; some studies have suggested that the associations of smoking with all-cause CRC mortality were higher among patients with microsatellite-stable or MSI-low tumors, whereas others have reported a similar association with MSI-high tumors [4,13,24]. Data regarding genetic analyses of CRC specimens were not available in our database, which precludes the further exploration of the mechanisms underlying the association between cigarette smoking and an increased risk of CRC mortality.

Clinically, cigarette smoking is associated with later stages of CRC at diagnosis, which leads to a poorer prognosis and survival [34]. However, the increased risk persisted in our study despite matching for cancer stage. In addition to a proneoplastic effect, tobacco smoking constitutes a primary risk factor for cardiovascular and pulmonary diseases [32]. Therefore, smoking patients with CRC may incur additional risk or mortality from these causes. A prolonged induction period of more than 35 years is required for smoking to increase the risk of incident CRC [10,11,14,20]. The shorter duration of smoking associated

with an increased CRC mortality risk in our study may support the involvement of smokinginduced comorbidities.

Studies have indicated that colon and rectal cancer may have partly different etiologic pathways and should be considered to be two separate entities that differ in susceptibility to carcinogens [11,17]. However, no consensus has been reached regarding whether the risk of incident colon or rectal cancer is more strongly associated with smoking [1,10–12,16–18,20,21,27,35–39]. Similarly, the association between smoking and CRC subsite mortality has been the topic of debate; several studies have reported that smoking was more significantly associated with a worse survival in patients with colon cancer than in those with rectal cancer [4,13,24]. However, others have reported a similar association between smoking and colon and rectal cancer mortality [9]. In our study, smoking was associated with an increased risk of mortality in patients with colon but not rectal cancers, which implies a higher susceptibility in patients with colon cancer. A significant dose– response relationship also supports the stronger association between smoking and colon cancer mortality.

The most pronounced advantage of our study was the sample size of the nationwide population-based cohort study. The number of events regarding the association between smoking and CRC mortality in our study was greater than those in previous studies [4,9,11,13,19]. We chose ever smoking as the main exposure risk because the effects of smoking may persist after changes in smoking behavior [4,39]. Studies have also reported a similar CRC risk for former and current smokers [15,21]. To obviate the influence of potential confounders, we performed propensity score matching to generate the study cohort. We further adjusted for factors, such as the alcohol consumption and body mass index, because they have been closely associated with both smoking and cancer risk [5,9,10]. The results from this and other studies demonstrate that the effects of smoking in CRC were much smaller than those in cancers of the respiratory and upper gastrointestinal tracts [16]. Nevertheless, quitting smoking for at least 20 years may still significantly reduce the risk of CRC incidence and adverse outcomes, suggesting smoking as a potentially modifiable risk factor of CRC prognosis [4,9,13,18].

It might seem peculiar that patients in the highest smoking amount category did not always have an increased risk of mortality in this study, including various subgroup analyses, as did patients in the highest smoking duration category. This might be attributable to the lower thresholds of smoking duration and amount required to increase the risk of CRC mortality in our study population. Furthermore, we speculate that the differential relationship between smoking intensity or smoking duration and CRC survival may also contribute to this phenomenon, that is, a threshold relationship for smoking intensity and a dose–response relationship for smoking duration. The relatively small number of patients in the highest smoking amount category in the whole cohort and in subgroups might also be accountable. Lastly, it might be postulated that the impact of the smoking duration on the risk of CRC mortality outweighs that of smoking intensity in our study population.

The limitation of this study stemmed largely from the use of administrative databases. The self-reported and retrospective collection of information on smoking and other variables was prone to recall and reporting biases. Data regarding family history of CRC, dietary information, physical activity, CRC screening, and use of cyclooxygenase inhibitors were not comprehensively recorded. Most importantly, other measures of smoking behavior, such as age at initiation, cumulative cigarette pack years, and passive smoking, were not collected. These factors may compromise the accuracy of the analysis. Furthermore, although we performed propensity score matching and adjusted for multiple covariates associated with smoking and CRC prognosis, the possibility of residual confounding cannot be excluded. The misclassification of anatomical subsites of CRC may have occurred, especially for tumors located in the junction of the sigmoid colon and the rectum. Finally, data regarding the molecular derangements of cancer were not available. These factors preclude a further detailed analysis of the differential effects of smoking on subsite CRC mortality.

#### **5. Conclusions**

Our study demonstrated that cigarette smoking was associated with a significantly, though moderately, increased risk of mortality in Asian patients with CRC. The smoking status can plausibly be considered in the risk stratification of CRC, and smoking cessation can be incorporated into comprehensive treatment planning for patients with CRC.

**Author Contributions:** Conceptualization, Y.-M.H., P.-L.W., C.-H.H. and C.-C.Y.; Methodology, C.-H.H. and C.-C.Y.; Validation, Y.-M.H., P.-L.W. and C.-C.Y.; Formal Analysis, C.-H.H.; Writing— Original Draft Preparation, Y.-M.H., P.-L.W., C.-H.H. and C.-C.Y.; Writing—Review and Editing, Y.-M.H., P.-L.W., C.-H.H. and C.-C.Y.; Supervision, P.-L.W.; Project Administration, P.-L.W. and C.-C.Y.; Funding Acquisition, P.-L.W., C.-H.H. and C.-C.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Health and Welfare Surcharge of Tobacco Products, grant numbers MOHW107-TDU-B-212-114020, MOHW108-TDU-B-212-124020, MOHW109-TDU-B-212- 134020, and MOHW110-TDU-B-212-144020.

**Institutional Review Board Statement:** This study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Research Ethics Committee of Chi Mei Hospital (IRB no. 10702-E04).

**Informed Consent Statement:** Requirement for patient informed consent was waived by the Research Ethics Committee of Chi Mei Hospital.

**Data Availability Statement:** The data sources were the Taiwan Nation Health Insurance Database and Taiwan Cancer Registry. The data are available with permission from the Taiwan Health and Welfare Data Science Center (https://dep.mohw.gov.tw/DOS/np-2497-113.html, accessed on 16 November 2021). Restrictions apply to the availability of these data, which were used under license for this study.

**Acknowledgments:** We are grateful to the Health Data Science Center of National Cheng Kung University Hospital for providing administrative and technical support. The authors also thank Yu-Cih Wu and Yi-Chen Chen for their assistance in advancing this project.

**Conflicts of Interest:** The authors declare no conflict of interest.

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