Serum CA242: a biomarker for diagnosis, progression, and metastasis in multiple tumors

CA242 is a classic biomarker used for diagnosing digestive tract tumors, especially pancreatic cancer. However, CA242 serum levels in some tumor patients and what might inuence these levels remain unknown or uncertain. This study aimed to reveal the pancancer landscape of serum CA242 levels and identify some inuencing factors.


Patient characteristics
The serum CA242 levels of 37,493 patients with 35 types of tumors and 880 healthy controls were statistically compared and analyzed, and the results are shown in Table 1. To visualize the results, we constructed Figure 1 and ranked all the diseases from highest to lowest according to the median CA242 level.
The mean and median serum CA242 levels of most patients with malignant disease were signi cantly higher than those of the healthy controls, and the serum CA242 levels of all the patients with malignant digestive tract malignant tumors were increased. Compared to the healthy controls, the tumor patients with the most signi cantly increased median levels were those with pancreatic cancer (8.12-fold), gallbladder cancer (5.77-fold), or cholangiocarcinoma (4.78-fold). However, statistically, the patients with fallopian tube cancer, lymphoma, penile cancer, leukemia, benign colorectal neoplasms, or thymic cancer were not signi cantly different from the healthy controls ( Figure 1 and Figure 2).

CA242 in different metastatic situations
Serum CA242 levels varied widely in most diseases; for example, the median value for colorectal cancer was 8.39, but the quartile range was from 4.78 to 497.4 (Table 1 and Figure 1). We speculated that a factor such as the progression of the disease might affect the levels, so we divided the tumor patients into three groups according to their metastatic situation: no metastasis (NM), LNM, and DM.
We found that the mean and median serum CA242 levels were increased in most tumor patients who exhibited LNM or DM compared to those who exhibited NM. The diseases that showed the most signi cant increases in the serum CA242 level were cholangiocarcinoma [median (quartile) for NM: 17 We constructed ROC curves for the serum CA242 values of the patients with each type of tumor versus that of healthy controls. The AUC value positively correlated with metastasis in most tumors. In patients with cholangiocarcinoma with LNM or pancreatic cancer with DM, the AUC of CA242 was greater than 0.9, indicating that CA242 has a high diagnostic value. In 6 diseases including cholangiocarcinoma, gallbladder cancer, periampullary carcinoma, colorectal cancer, gastric cancer, and pancreatic cancer, when LNM or DM appeared, the AUC values were all signi cantly higher than 0.7, and DM produced a larger increase than LNM (Table 2).

CA242 in different clinical stages
We analyzed the serum CA242 values of 11 types of tumors in 2,500 patients who had certain clinical staging information.
We found that the mean and median levels of serum CA242 in most tumor patients increased with stage progression and were almost all higher than those in healthy controls. The median CA242 level in stage IV gastric cancer was 22.87 (7.33, 84.00), and that in stage IV colorectal cancer was 20.48 (7.74, 61.255). Both of these values were approximately ve times higher than the value for the healthy controls [4.32 (2.46, 7.26)]. Interestingly, The means of some tumors in stage IV, including lung cancer (31.65±67.96), ovarian cancer (34.37±69.27) and cervical cancer (26.04±47.08), were approximately 5-6 times higher than the mean of the healthy controls (5.58±4.56), but the corresponding medians were only approximately two times higher. However, the medians in ovarian cancer and lung cancer did not change signi cantly between stages II and III. No signi cant differences were observed in the following diseases: nasopharyngeal carcinoma in stage I or II, laryngeal cancer in stage I or II, bladder cancer, renal cancer, esophageal cancer, and endometrial cancer (Table 3).
Then, the ROC curves of the CA242 level in each tumor stage relative to that in the healthy controls was calculated. The analysis showed that the AUC of CA242 gradually increased with progression in most tumors. Among these tumors, colorectal cancer, gastric cancer, cervical cancer, and lung cancer were particularly remarkable. Especially in stage IV, the AUC values of these four tumors were greater than 0. 8 Figure 3). CA242 in different metastatic sites Based on the above results, we noticed that serum CA242 levels exhibited a signi cant change with DM and had the capacity to predict metastasis in some tumors. Therefore, we wanted to study whether there is a correlation between patient serum CA242 levels and sites of metastasis. Therefore, we divided 3,953 patients with one of ve cancers who had de ned metastatic site information into group NM and group DM.
We found that the mean and median values of the serum CA242 level in DM patients in most diseases were higher than those in NM patients and healthy controls, but there were no signi cant differences among the patients with different metastatic sites within each tumor type (Supplemental Table 3). We speculated that this lack of difference was due to the small sample sizes for some metastatic sites and the large variation range of CA242. Therefore, we calculated the median fold change in DM patients relative to NM patients for every tumor type and the log2 (fold change) value of the serum CA242 level for each metastatic site and found the following results: multiple metastases: 1.228 (-0.1189, 3.079), liver metastasis: 1.071 (-0.1195, 3.059), bone metastasis: 0.7286 (-0.2386, 1.979), brain metastasis: 0.6536 (0.2648, 2.401), and lung metastasis: 0.4921 (-0.3371, 1.676). There were signi cant differences among the metastatic sites other than the brain (Table 4).

Discussion
Although CA242 has been identi ed and used in clinical practice for some time, this is the rst study to pro le the levels of this biomarker in 35 different tumors in a large number of patients. Among the 35 tumor types, the serum CA242 level changes in 23 tumor types had never been reported before. We also found that serum CA242 levels in patients with metastatic tumors might be related to the target organs containing metastases for the rst time.
We found that CA242 levels were elevated in a variety of digestive system cancers, including pancreatic cancer, gallbladder cancer, cholangiocarcinoma, colorectal cancer, and gastric cancer, which was consistent with previous reports. The AUC values of the ROC curves for pancreatic cancer, gallbladder carcinoma, and cholangiocarcinoma were all greater than 0.8, but only pancreatic cancer has been studied in detail [4,10,13,17,22,[25][26][27]. Previous studies have shown that CA242 alone or in combination with other biomarkers can predict the diagnosis, progression, and prognosis of pancreatic cancer [4, 7, 10, 12-16, 23, 24]. Pancreatic cancer has been the leading clinical application for CA242 because of its excellent performance in this cancer. Some small-scale studies have investigated CA242 in gallbladder carcinoma and cholangiocarcinoma [7,14,25,28], but due to the sample size or quality of these studies, the credible AUC values for these two carcinomas are still unknown. Therefore, our ndings indicate that CA242 is also suitable for use as a biomarker for cholangiocarcinoma and gallbladder cancer, whose AUCs were 0.8145 (0.7737, 0.8553) and 0.8349 (0.8157, 0.8542), respectively ( CA242 levels in patients with gastric cancer differed between stage IV and non-stage IV[20], but alone, CA242 was not a good predictor of LNM [21]. However, in a study by Fangxuan Li et al.[31], the mean value of 29.84±83.54 in gastric cancer was not much different from our data, but their AUC for gastric cancer relative to healthy people was 0.809, which is much higher than the value of 0.6839 (0.604, 0.8202) found in this study (Table 1). We suggest that a possible explanation may be that their sample size is too small and that their mean lower than the control is 3.85 ± 3.13. A surprising nding was that the CA242 level in patients with gastric cardia adenocarcinoma was not signi cantly different from that in gastric cancer patients.
In this study, the mean value in lung cancer was 18.49±45.97 (Table 1), but Huaiqian Dou et al. [10] reported that the mean in lung cancer was 9.34±13.67. In contrast, Xiaochuan Wang et al. [32] reported that it was 28.39±51.91. These differences might be caused by a sampling error, detection kit choice, and instrument differences. However, the results we reported have the largest sample size and therefore higher credibility. The mean value in ovarian cancer was 19.52±42.27 (Table 1), which was slightly higher than the previously reported value of 11.40±16.88 [10], and the mean in cervical cancer was 11.54±23.97, which was very similar to the previously reported value [10]. The levels in nasopharyngeal carcinoma, craniofacial malignant tumor, endometrial cancer, breast cancer, renal cell cancer, liver cancer, esophageal cancer, and prostate cancer patients were signi cantly higher than those in healthy controls ( Table 1), indicating that CA242 has potential clinical value in these diseases. Although mediastinal cancer, testicular cancer, ureteral cancer, and neuroendocrine cancer patients had higher mean and median values than health controls (Table 1), the sample size was too small to identify signi cant differences.
The CA242 levels in colorectal cancer and gastric cancer patients increased not only with the appearance of LNM or DM but also with advancement from stage I to IV[18, 20-22, 26, 30]. The results mentioned above are consistent with the trends in a previous study, and we also found that CA242 was able to distinguish colorectal cancer and gastric cancer patients from healthy controls ( Table 1). The AUC values for diagnosing stage IV disease reached 0.85 or higher and, for DM, reached 0.8299 (0.8117, 0.8481) and 0.7359 (0.6955, 0.7764) in colorectal and gastric cancer patients, respectively (Table 2 and   Table 3). In patients with pancreatic cancer, cholangiocarcinoma, gallbladder carcinoma, lung cancer, cervical cancer, ovarian cancer, periampullary carcinoma, bladder cancer, breast cancer, or endometrial cancer, serum CA242 levels were positively correlated with LNM and DM (Table 2). Some diseases, such as pancreatic cancer, have been reported, but the rest are reported here for the rst time. Meanwhile, we noticed something different that in certain tumor types, especially cholangiocarcinoma, CA242 levels in LNM were signi cantly higher than DM (Table 2). Numerous studies have shown that LNM is one of the most prominent prognostic factors associated with poor prognosis for cholangiocarcinoma patients[33].
We considered that lymph nodes might be a paradise for cholangiocarcinoma cells so that LNM led to higher CA242 compared to DM. There were no signi cant differences in CA242 levels between patients with laryngocarcinoma, esophageal cancer, nasopharyngeal cancer, or renal cancer metastasis and those with NM nor were there different among patients with various stages of disease for these cancers (Table 2). Furthermore, such a phenomenon had caught our attention, that was the means of lung cancer, ovarian cancer and cervical cancer in stage IV were 5-6 times higher than healthy controls, but their medians merely rose no more than two times. Something special that happened to a handful of these stage IV patients was supposed to cause their CA242 zooming up and distributing skewed in the whole stage IV sample, so there was a difference between mean and median in the three tumors. In future studies, we will continue to pay attention to this phenomenon to make further discoveries.
Among thyroid cancer, prostate cancer, and craniofacial malignant tumor patients, the level of CA242 increased with metastasis, but the differences were not signi cant ( Table 2). While endometrial cancer and bladder cancer showed signi cant decreases in the sample, although the CA242 level showed an increasing trend with advancing clinical stage, the data showed no signi cant differences ( Table 3). The remaining tumor sample sizes were too small to draw valid conclusions. According to whether the CA242 level increased with metastasis or stage advancement in the tumors listed in Tables 2 and 3, we identi ed tumors as "CA242 progression-positive tumors". In these tumors, the combination of other tumor markers and CA242 could be further studied to improve the accuracy of the prediction of tumor progression. We speculate that some commonalities exist among these tumors, such as mutations in similar tumor driver genes[34], similar embryonic developmental origins, and microenvironments. Further research is needed to con rm these ndings, which may have the potential to facilitate the diagnosis and treatment of tumors.
Furthermore, we also found that in all of the cancers listed in Table 4, the increase in the serum CA242 value for multiple metastases compared to NM was the largest, followed by the increases for liver metastasis and bone metastasis. Some studies have shown that the prognosis of gastric cancer with liver and bone metastasis is worse than that of any other subtype of gastric cancer, and the two organs even account for most metastatic sites [35,36]. Interestingly, we nd that similar situation also exist in colorectal cancer, lung cancer, pancreatic cancer, breast cancer and other tumors [37][38][39][40].We deem that the liver and bones may have microenvironments conducive to the synthesis and release of CA242 molecules in the ve "CA242 progression-positive tumors" or that the liver and bones enhance metastatic tumor cell growth so that more tumor cells produce more CA242, resulting in elevated levels. Therefore, CA242 may be valuable in predicting the progression of "CA242 progression-positive tumors". Moreover, studying the molecular mechanisms underlying the elevated CA242 levels may produce an understanding of the mechanisms of tumor metastasis.
However, not all malignant tumor patients' serum CA242 levels were greater than those of healthy controls. For example, the sample sizes for leukemia, laryngocarcinoma, skin cancer, and penile cancer were not small, but there were no signi cant differences between these patients and the healthy controls in regard to the median levels ( Table 1). This nding indicated that serum CA242 levels are not suitable for diagnosing all diseases, but CA242 may be used to identify nonprimary tumors at the same site, that is, metastatic tumors with increased CA242 expression. Moreover, the median serum CA242 values of the two precancerous lesions in this study, benign colorectal neoplasms and cervical intraepithelial neoplasia, were similar to the median value of the healthy controls, and there were no signi cant differences. However, the median CA242 level in benign colorectal neoplasms was less than half of that in colorectal cancer, but the difference was not signi cant, which is probably caused by the small sample size for benign colorectal neoplasms. By increasing the number of samples, we might nd a difference between these two precancerous lesions and a new marker to identify benign colorectal neoplasms and colorectal cancer, as is done for cervical cancer and cervical intraepithelial neoplasia.
This study has several limitations. First, the sample size is relatively insu cient. Although the total sample size was more than 35,000, there were 35 different kinds of diseases, so each disease included only 1,000 patients on average. Thus, some tumors, such as colorectal cancer and liver cancer, were numerous, with more than 4,000 cases. However, some had many fewer cases, such as mediastinal tumor, ureteral cancer and fallopian tube cancer, which had only 10 cases. The reasons for this situation are not only the disparity in the incidence rates of different diseases but also the previous understanding of the diagnostic role of CA242. CA242 is considered to be a diagnostic marker for digestive system tumors [10,13,22, 26], especially pancreatic cancer, so measuring CA242 levels is not an appropriate choice when a doctor suspects a patient may develop other tumors. Second, in the analysis of individual diseases, the subpopulations are not su ciently accurate. In this study, we strati ed subpopulations according to the metastatic status, clinical stage, and metastatic site of the tumors.
Although some differences and factors that may affect the level of CA242 were revealed, we still did not nd an excellent strati ed method that could make the distribution concentrated and failed to nd further accurate factors that in uenced CA242 levels. Third, the collection of patient clinical information is relatively incomplete. For example, only some patients had accurate metastasis and clinical staging information, and the data of perioperative period have not yet been collected.
Because we gathered no staging classi cation information for some tumor patients, such as those with pancreatic cancer, cholangiocarcinoma or gallbladder cancer, we missed the opportunity for further analysis of the diseases that most commonly use CA242 in the clinic. Lack of perioperative period data also makes it impossible to discover the impact of CA242 in the choice of surgical resectability in these tumors. Fourth, this was a single-center, retrospective study. Therefore, the numbers of patients in each stage and metastatic status were very different, which may lead to biased conclusions. In addition, the CA242 values for different stages and metastatic states belonged to different patients. In other words, this study had neither a paired design nor a longitudinal design so some confounding factors may have in uenced the conclusion of the study.
In this study, we analyzed serum CA242 levels in 35 types of neoplastic disease patients and healthy controls and found that these levels remained unchanged in benign tumors and increased most in malignant tumors, especially pancreatic cancer, gallbladder cancer, cholangiocarcinoma, and periampullary carcinoma. The CA242 levels of these malignant digestive system tumors were signi cantly higher than those of other tumors and were valuable for patient diagnosis. Moreover, the level of CA242 was also related to metastasis and clinical stage and was able to predict the progression of some tumors.

Conclusions
In conclusion, CA242 can not only serve as a diagnostic biomarker for malignant digestive system tumors, such as pancreatic cancer, but also predict the progression, stage, metastasis, survival, prognosis, etc. of tumors or play a role in tumors that have not received clinical attention and are awaiting further research.

Declarations
Ethics approval and consent to participate This study was approved by the Ethics Committee of the First A liated Hospital of the Army Medical University, PLA (the approval number is KY201912), and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Consent for publication
Not applicable

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests