The Association of Serum 25-Hydroxyvitamin D and Carcinoembryonic Antigen in Locally Advanced Colorectal Cancer With Neoadjuvant Chemoradiotherapy

Backgrond We aimed to describe 25-hydroxyvitamin D [25(OH) D] and carcinoembryonic antigen association levels in patients with locally advanced colorectal cancer (LARC) treated with neoadjuvant chemoradiotherapy (nCRT). Methods Between July 2012 and September 2018, the records of 92 stage II/III colorectal cancer patients with treated with nCRT followed by radical surgery were reviewed retrospectively. Enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay was used to analyze the patient’s carcinoembryonic antigen (CEA) and 25(OH) D concentrations in serum. Results The serum CEA in was lower while the serum 25(OH) D from pre-nCRT patients was signicantly higher than post-nCRT patients. Our results showed that the CEA and level is a risk factor of LARC.

25(OH) D and carcinoembryonic antigen (CEA).Plasma 25 (OH) D levels is a prognostic biomarker with low 25(OH) D associated with poorer survival.Thus, serum 25(OH) D and CEA levels might be of value in evaluating the pathogenesis and risk of LARC in the future.Moreover, serum CEA or 25(OH) D levels were associated with patient's clinical and pathological features providing data for risk and prognostic prediction.

Background
Colorectal cancer (CRC) is a common type of cancer worldwide [1].In China, the estimated new cases and deaths of CRC were 376300 and191000, respectively, in 2015 [2].Neoadjuvant chemoradiotherapy (nCRT) has been used as a consensus-based treatment modality for stage II/III colorectal cancer [3].The move has heightened sphincter preservation rates and lower local recurrence rates, and also down staging of the disease.Accumulating evidence indicates that high levels of circulating vitamin D are an anti-cancer factor, which may reduce the risk of CRC [4][5][6].
In the human body, vitamin D participates in the metabolism of calcium, phosphorus and other substances, and it is believed to be highly associated with cancer as well as other chronic diseases [7].
Accumulating data suggest that vitamin D de ciency is highly prevalent among patients with CRC.In the liver, vitamin D is converted by 25-hydroxylase into 25 hydroxyl vitamin D3 (25(OH)D3), the main circulating form of vitamin D and the most reliable measurement of an individual's vitamin D status [8].
The neoadjuvant chemo radiotherapy resistance contributes to substantial mortality in advanced colorectal cancer, it would be representatives for alternative or rigorous treatment approaches.So, identifying predictive biomarkers of response in advanced following nCRT would be of signi cant clinical relevance.Among the biomarkers, Carcinoembryonic antigen (CEA) is the most useful predictor to estimate the sensitivity of nCRT in colorectal cancer [9].So far, the expression levels of 25(OH) D and CEA in locally advanced colorectal cancer, and their association with advanced colorectal cancer pathogenesis and risk are still ambiguous.In this study, we analyzed the serum 25(OH) D and CEA levels in pre-and post-neoadjuvant chemo radiotherapy locally advanced colorectal cancer patients and correlated them with clinical features and the risk of locally advanced colorectal cancer.

Patients
Ninety two patients diagnosed with locally advanced colorectal cancer were enrolled consecutively in this study in a period of time from July 2012 and September 2018.The diagnosis was made by pathologic examination.During hospitalization; all patients had no outdoor activity and were provided diet without calcium or vitamin D supplements.75 healthy individuals, aging from 45 to 77 years old, who had regular diet without calcium or vitamin D supplements before blood drawing, were selected as controls with matched ages and genders to the patient group.The study was approved by the Institutional Review Board of the Changzhou Cancer Hospital Autonomous Region for the use of human materials.

Blood sample collection
Fasting blood samples were obtained either the day before nCRT or other treatments (baseline) and on day 14 after nCRT, respectively, using the serum separation tube (BD healthcare).All patients with locally advanced colorectal cancer had paired pre-nCRT and post-nCRT blood samples.The blood samples were transported to the Department of Laboratory Medicine within 30 minutes of collection.The samples were then centrifuged at 1500 g at 4°C for 10 minutes; the serum was collected into a 2 mL of centrifuge tube and stored at − 80°C until analysis.

Enzyme-linked immunosorbent assay
CEA was measured by using the Quantizing ELISA Human CEA Immunoassay kit from R & D Systems, in following the manufacturer's instructions.In brief, 100µL of Assay Diluent RD1-19 was added to each well in the microplate; then, 50µL of standard, control, or sample were added per well and covered with the adhesive strip to incubate for 1 hour at room temperature on a horizontal orbital microplate shaker; all the liquid was aspirated and washed by adding 400µL Wash Buffer for 4 times; after the last wash, inverted and blotted against the plate through clean paper towels to drain he remaining wash buffer; added 200µL of Human CEA Conjugate to each well and covered with a new adhesive strip to incubate for 2 hours at room temperature on the shaker; repeated the aspiration and wash; then added 200µL of Substrate Solution to each well to incubate for 30 minutes at room temperature on the benchtop by protecting from light; following this step, 50µL of stop solution was added to stop the color development; and nally, measured the plate immediately on a microplate reader at OD 450 nm.

Chemiluminescence immunoassay
In clinical laboratory, chemiluminescence immunoassay is widely used to measure serum vitamin D (VD) because of its automatic capability with clinical satisfaction.In this study, serum VD was quantitatively analyzed on a Roche cobas E601 electrochemiluminescence immunoassay platform using a Roche cobas Elecsys Vitamin D total (25Hydroxyvitamin D, REF 05 894 913 190, Roche Diagnostics GmbH) assay kit.

Statistical analysis
IBM SPSS Statistical 24.0 (SPSS Inc Chicago) was performed for statistical analysis.Differences in basic characteristics of patients were evaluated by using independent samples t test.Paired samples T test was performed to compare the difference in each parameter of paired pre-and post-nCRT serums or 25(OH) D levels.Moreover, Pearson's correlation analysis was applied to evaluate the association between serum CEA and 25(OH) D levels in LARC.The pre-and post-nCRT 25(OH)D and pre-and post-nCRT CEA (≥ median and < median) levels were strati ed by using conditional logistic regression (multivariate analysis) to evaluate whether serum CEA or 25(OH)D is a risk factor for LARC, which estimated odds ratios (OR) and 95% con dence intervals (CI).A P-value of < 0.05 was considered as statistically signi cant.Serum 25(OH) D and CEA levels were correlated with clinical and pathological features including age, gender, lesion site, tumor size, lymph node involvement,CRT regimen.

Ethics statement
The study was approved by the Institutional Review Board of the Changzhou Cancer Hospital Autonomous Region for the use of human materials.

Correlation of serum CEA and 25(OH) D levels with clinical-pathological features in LARC patients
The correlation of serum CEA and 25(OH) D levels with clinical pathological features from 92 patients with LARC were summarized as shown in Table 1.Of 92 patients with LARC, there were 39 females and 53 males with ages ranging from 35 to 82 years (a median age of 61 years).In this study, colon was the main lesion site of the tumor (49/92).
According to the parameters we collected, we divided each parameter into two groups, which was analyzed through univariate analysis (displayed in Table 1).In independent samples test, our data showed that the groups, tumor size 5.0 cm, lymph node involvement, that has no distinctly lower serum post-nCRT CEA levels than the groups(p 0.05).As for the levels of pre-nCRT serum 25(OH) D, males, tumor size < 5.0 cm, lymph node invasion groups were signi cantly higher (P = 0.005, P = 0 .008,P = 0 .049,respectively).While with post-nCRT 25(OH) D levels, males, lymph node invasion, and tumor size < 5.0 cm had signi cantly higher levels (p < 0.05, respectively).In paired samples test, the post-nCRT CEA concentration of patients were signi cantly lower than the pre-nCRT CEA levels except the group of lymph node invasion.Most surprisingly, in the standard radiotherapy and chemotherapy regimens, Capecitabine did not change the 25(OH) D levels before and after neoadjuvant chemo radiotherapy.2 shows that the serum CEA in pre-and post-nCRT LARC patients was signi cantly higher than the controls (P < 0.001 for both).While serum 25(OH) D in both pre-and post-nCRT LARC patients were signi cantly lower than the controls (P < 0.001 for both).Moreover, the CEA levels in pre-nCRT LARC patients was signi cantly lower than the post-LARC levels (P < 0.005).However, the level of pre-nCRT 25(OH) D was signi cantly higher than post-nCRT 25(OH) D (P < 0.005).

Serum CEA and 25(OH) D levels and the risk of
So as to understand whether serum CEA and 25(OH) D is a risk factor for LARC, multivariate analysis and conditional logistic regression were performed and the results are displayed in Table 3.The results showed that the pre-nCRT CEA level is a risk factor for LARC (OR = 13.145,CI = 1.71-110.31,P = 0 .012),while the post-nCRT 25(OH) D level is a protective factor (OR = 0.083, CI = 0.01-0.84,P = 0.040).

Correlation between serum CEA and 25(OH) D levels
Our research results demonstrated that there was a weak correlation between both pre-nCRT CEA and post-nCRT CEA with 25(OH) D levels (Table 4, Pearson − .126and .154,P = .265and .171,respectively).

Discussion
Several studies have predicted the sensitivity of nCRT in colorectal cancer.Imaging modalities that were used for evaluation included CT and ERUS [10].The frequency of vitamin D insu ciency in patients with locally advanced CRC has not been reported in larger studies.Several studies suggested that the expression levels of four biomarkers (p53, VEGF, p21, and Ki67) were signi cantly correlated with PCR to nCRT (96.3 %).However, this scoring system showed only 46.3% speci city.Her-2, pre-CEA, and post-CEA were also considered as predictors and of their disadvantages [11,12].Altered CEA was proved more signi cant to re ect the sensitivity of colon cancer to chemotherapy than pre-or post-CEA [13,14].So,ΔCEA was utilized as a positive control to compare with 25(OH) D level in our study.There is a hypothesis that alteration of vitamin D receptor may change transcriptional control of 1, 25(OH) 2D3 and impact anti-tumor effect of vitamin D [15].These results suggest that vitamin D plays a role in inhibiting tumorigenesis of CRC.Our results showed that both serologic levels of pre-and post-nCRT CEA in LARC patients were distinctly higher than the healthy controls (Table 2).We also found that pre-nCRT CEA levels was a risk factor for LARC (OR = 13.145,CI = 1.71-110.31,P = 0 .012,Table 3, respectively).
In our research, the serum concentration of pre-nCRT CEA was much lower than the post-nCRT level ( Table 2), while the concentration of pre-nCRT 25(OH)D was much higher than the post-nCRT level (P < .001,Table 2).It seems that CEA and 25(OH) D had an inverse association with the disease progression.However, there was no signi cant correlation between these two markers in the serum by Pearson's correlation analysis (Table 4) in our study.Perhaps the small sample volumes reduce the relationship between CEA and 25(OH) D. Due to the limitations of patient management in our hospital; we were unable to measure the serum CEA and 25(OH) D levels at multiple time points in this study.
Many studies found that there is an inverse association between serum 25(OH) D and the risk of CRC [16-18].We found that both serologic levels of pre-and post-nCRT 25(OH) D in patients with LARC were signi cantly lower than in healthy individuals.Our research of univariate analysis showed that the serum concentration of pre-and post-nCRT 25(OH) D in male patients and tumor size < 5.0 cm were signi cantly higher (Table1).A potential explanation could be that a more advanced stage of disease and/or larger tumor size is accompanied with more in ammation, which may lead to lower 25(OH) D3 levels [19,20].It could be that the absorption of vitamin D from diet and supplements is impaired due to subclinical mucositis induced by chemotherapy [21,22].Andersen et al found that the inverse association between serum 25(OH) D and CRC was more prominent in females than males [6].A meta-analysis including 1248 cases of CRC suggested that 25(OH) D had stronger inverse associations with colon than with rectal tumors [23].However, in our study, the serum concentration of pre-and post-nCRT 25(OH) D in rectal and colon tumors were not statistically signi cant.The serum concentration of pre-and post-nCRT CEA in groups of tumor size 5.0 cm and lymph node invasion patients not signi cantly reduced.
A report suggests that low vitamin D levels were found in most cases of breast cancer with advanced tumor stage, large tumors, and positive lymph nodes [24].In line with our results, the study of Fakih and colleagues observed a threefold higher risk of having vitamin D de ciency in CRC patients [25].In line with our results, the study of Isenring and colleagues also reported a decrease in 25(OH) D levels in cancer patients receiving chemotherapy [26].The lower 25(OH)D3 levels and/or the steeper decrease in 25(OH)D3 levels in patients receiving chemotherapy can be induced by changes in lifestyle due to chemotherapy or by chemotherapy directly [27].The study of Churilla et al., found a higher prevalence of vitamin D de ciency in stage III of disease [28].But, Some studies show that no difference in the 25(OH) D levels from baseline to six-month follow-up between patients who did and did not receive chemotherapy (P = 0.26), suggesting that chemotherapy may not have the deleterious effects on 25(OH)D levels that we ascribed to increased photosensitivity and potential for gastrointestinal toxicities [29].And furthermore, Song et al. have found that the associations between vitamin D de ciency and CRC in a large nested case-control study remain valid after adjustment for in ammatory markers [30].

Conclusions
In conclusion, our study found that there was a relationship between serum 25(OH) D and CEA in LARC.Thus, serum 25(OH) D and CEA levels might be of value in determining the pathogenesis and risk of LARC and requires further study. Declarations

Table 1
Correlation of serum CEA and 25(OH) D with clinical-pathological features a represents the comparison of intra-parameter.P r epresents the comparison of CEA and 25(OH) D in pre-and post-nCRT LARC.Abbreviations: Post-nCRT, post-neoadjuvant chemoradiotherapy; Pre-nCRT, pre-neoadjuvant chemoradiotherapy.*Means P < 0.05 *Means P < 0.05 3.2 Serum CEA and 25(OH) D levels in pre-and post-nCRT LARC patients

Table 2
Serum CEA and 25(OH) D levels in pre-and post-neoadjuvant chemoradiotherapy LARC patients and controls

Table 3
Evaluation of pre-and nCRT serum CEA and 25(OH) D as a risk factor in LARC patients *Means P < 0 .05.

Table 4
Comparison between serum CEA and 25(OH) D in LARC by Pearson's correlation analysis