C-Reactive Protein/Albumin Ratio Is a Powerful Prognostic Indicator for Survival in Surgically Resected Gastrointestinal Stromal Tumors: A Retrospective Cohort Study

Background Systemic inammation and malnutrition may promote tumor progression. C-reactive protein/albumin ratio (CAR) is linked with poor long-term survival of several malignant tumors. Purpose To explore the predictive value of CAR in gastrointestinal stromal tumors (GISTs). Methods A retrospective study was conducted on 325 patients with GIST who underwent radical surgery from 2009 to 2018. The cut-off point of CAR was set using X-tile software. Kaplan-Meier method and multivariate Cox regression model were used to study the prognostic value of CAR. The time-dependent receiver operating characteristic curve(tROC) was drawn, and the prognostic accuracy of CAR, Glasgow prognosis score (GPS), and NIH risk classication was compared by the area under the curve (AUC). Results


Background
Gastrointestinal stromal tumors (GISTs) are the most frequent soft tissue sarcomas with an annual incidence in the United States of approximately 7 per million population [1]. The incidence has risen slightly over the last decade [1]. In China, due to the inaccuracy of provincial GIST registration data, the National Cancer Center's annual report does not include the incidence of GIST. However, the incidence is estimated to be between 4.3 and 21.1 per million population [2]. Surgery and targeted agent therapy have made progress, but the prognosis of GIST patients is still weak due to early recurrence or distant metastasis [3,4]. Therefore, identifying biomarkers that can be used to predict adverse surgical and oncological outcomes based on patient-speci c risk will be essential for guiding surgery and optimizing targeted drug therapy for GIST.
Extensive studies have shown that in ammation and malnutrition may promote the occurrence, progression, and metastasis of malignant tumors [5][6][7]. Several studies have identi ed that in ammatory markers based on white blood cells and acute-phase proteins are related to poor prognosis in various tumors [8][9][10][11]. In recent years, the role of C-reactive protein (CRP) and albumin in the interaction network between systemic in ammatory response and cancer has attracted increased attention [12][13][14]. Markers based on CRP and albumin, such as the Glasgow prognosis score (GPS) and its variants, are correlated with cancer patients' long-term survival, including GIST [15][16][17]. The CAR, a combination of serum CRP and ALB, has been used to assess cancer patients' immunonutritional status and has recently been identi ed to be linked with poor long-term survival of various malignant tumors [18][19][20]. Accordingly, CAR may have a potential impact on predicting the long-term survival of GIST. However, the predictive value of CAR on the survival of patients with GIST after curative resection remains poorly understood.
The current research's primary aim was to investigate the relationship between preoperative CAR and the clinical outcome of GIST patients undergoing radical resection. Besides, we evaluated the correlation between CAR and CRP, ALB, GPS, compared their predictive ability, and nally investigated whether CAR combined with NIH risk strati cation system can improve its prognostic value.

Ethics statement
All participants obtained the written informed consent that their data was kept in the hospital data bank and used for research approved by the Ethics Committee of the Beijing Hospital, the National Geriatrics Center. The collection of data follows the Helsinki Declaration's principles, following existing national legislation, and in line with the principle of the protection of personal data.

Patients
From January 2009 to January 2018, a total of 325 patients with primary GIST who underwent open or laparoscopic surgery in the Department of Gastrointestinal Surgery of Beijing Hospital directly under the National Health Commission of China were considered to be included in this study.
The inclusion criteria were: (1) Pathologically diagnosed as GIST, with no distant metastasis; (2) underwent R0 resection (the surgical margin was negative); (3) ASA physical status before surgery is classi ed as I-III; (4) patients with detailed clinicopathological and preoperative serum laboratory data.
The exclusion criteria were as follows: (1) complicated with other primary malignant tumors; (2) infectious and non-cancerous in ammatory diseases; (3) Tumor rupture occurred during operation.

Data collection and de nition
The pathological diagnosis and risk assessment of GIST is based on the recommendations of the latest version of the China guidelines [21]. Patient characteristics assessed included age, gender, ASA PS classi cation, tumor size, tumor location, mitotic index, and NIH risk classi cation. The preoperative physiological status of the patients was evaluated by the ASA PS classi cation [22]. The results of serum CRP and albumin were obtained within one week before the operation. The CAR was calculated as follows: CAR= serum CRP (g/L)/albumin (g/L); The GPS was categorized as: score 2 represented the decrease of albumin(<35g/l) and the increase of CRP(>10mg/l); score 1 represented normal albumin(≥35g/l) and the increase of CRP(>10mg/l); and score 0 represented normal albumin (≥35g/l) and normal CRP(≤10mg/l)[23].

Follow-up
Routine follow-up included medical history (symptoms and physical examination), laboratory blood tests, abdominal and pelvic enhanced CT or MRI scans. Intermediate-or high-risk patients had CT or MRI scans every three months for three consecutive years, then every six months until the fth year, and then once a year after that. Low-risk patients had CT or MRI scans every six months for ve years. For patients who could not be followed up in the outpatient clinic, we conducted a telephone follow-up. The overall survival time (OS) was de ned as surgery date to the most recent follow-up or death. The last follow-up was in December 2020.

Statistical Analysis
The student t-test was used to evaluate the differences of continuous variables, while the Chi-square test was used to compare categorical variables. Survival analysis was performed by the Kaplan-Meier method with the log-rank test. Univariate and multivariate Cox proportional hazard models were performed to evaluate the impact of CAR on overall survival. The additive model was conducted to evaluate the interaction among subgroups to investigate whether other factors would affect the effect of CAR on OS. The prediction accuracy of different biomarkers was evaluated by time-dependent receiver operating characteristics (t-ROC) analysis. Statistical analysis was carried out with the SPSS version 26.0 (IBM Corporation, Armonk, NY, USA), the GraphPad Prism version 8.3.0 (GraphPad Software, Inc., San Diego, CA, USA), and the R software package version 4.0.2 (The R Foundation for Statistical Computing, Vienna, Austria; https://www.r-project.org/). Statistical signi cance was set at p < 0.05.

Clinicopathological characteristics
The detailed clinicopathological characteristics are summarized in Table 1 Using X-Tile program to calculate the best cut-off value We conducted an X-tile analysis for predicting ve-year OS and determined that the best cut-off value of the CAR was 0.55. Consequently, patients were separated into CAR-low (<0.55, n = 292) and CAR-high (≥0.55, n = 33) groups. We also determined that the best cut-off points of CRP and ALB were 18.20 and

OS in the CAR-low and CAR-high categories
The median follow-up period was 64 months, of which 14 patients (42.4%) died in the CAR-high category and 40 patients (13.7%) in the CAR-low category. GIST relapsed in 73 cases (22.5%), including 15 cases (45.5%) in the CAR-high category. Overall, the one-year, three-year, and ve-year OS rates were 93.8%, 83.9%, and 78.9%, respectively. In the Kaplan-Meier analysis, the CAR-high category (97.0%, 72.7%, and 57.0%, respectively) had a signi cantly shorter one-year, three-year, and ve-year OS than the CAR-low category in the total cohort (99.0%, 93.8%, and 88.0%, respectively; p<0.001) (Fig. 2a). Distinguished by the corresponding cut-off value (18.20 for CRP, 35.0 for ALB, and 1 for GPS), the ve-year overall survival rate in the CRP-high category, the ALB-low category, and the GPS 2 category (62.5%, 69.3%, and 50.4%, respectively) was signi cantly lower than that in the CRP-low category, the ALB-high category and the GPS 0 or 1 category (87.7%, 86.6%, and 86.7%, respectively; p<0.001) ( Fig. 2b-d).
Univariate and multivariate analysis of OS Univariate analysis indicated that ASA PS score, CRP, ALB, CAR, and GPS were signi cantly correlated with OS, and primary tumor size, tumor location, mitotic index, and NIH risk classi cation were also signi cantly correlated with OS ( Table 2). The multivariable analysis determined that the CAR (HR 2.598, 95% CI 1.385-4.874; p=0.003) was an independent prognostic factor for overall survival after radical resection, followed by NIH risk classi cation (p<0.001) ( Table 2).

Survival analysis of the interaction between CAR and clinicopathological factors
In order to further evaluate the prognostic value of CAR, we used univariate Cox regression for subgroup analysis to identify whether the effect of CAR on the overall survival of GIST patients was affected by other clinicopathological factors (Fig. 3). Therefore, in most subgroup analyses, there was a signi cant correlation between higher CAR and worse OS. Although there was no statistical signi cance in subgroup analysis between patients with primary gastric GISTs and patients under 60 years old, patients with high CAR tended to have a poor prognosis. However, this association does not apply to patients with a tumor size ≤5 cm, a mitotic index (per 50 HPF)≤5, and very low or low-risk classi cation.

Subgroup analysis according to NIH risk classi cation
In the intermediate or high-risk subgroup, the overall survival time of patients with high CAR was shorter (p = 0.001) (Fig. 4b). However, there was no signi cant difference in overall survival between high and low CAR patients in a very low-risk or low-risk subgroup (p = 0.628) (Fig. 4a).
Time-dependent ROC curve analysis to further evaluate the discriminating ability of CAR We conducted a time-dependent ROC analysis to evaluate the prediction accuracy of CAR, CRP, and GPS for OS (See Fig 5a). The AUC value of CAR at two year (0.601), and ve years (0.629) was higher than that of CRP (2 year:0.593, p=0.077; and 5 years:0.618, p = 0.132) and GPS (2 year:0.566 p =0.398, and 5 years:0.619, p =0.903), but the difference was not signi cant.
Finally, we evaluated whether the combination of CAR and NIH risk classi cation could improve predicting prognosis accuracy. As presented in Fig. 5b

Discussion
This study set out to assess the prognostic value of preoperative CAR in GIST patients who underwent radical resection and to compare it with proven markers of in ammation. The results show that preoperative CAR is an independent predictor for OS. Besides, CAR has better prognostic value in GIST patients with higher malignant phenotype. Moreover, the predictive ability of CAR is better than that of single systemic in ammatory markers such as CRP and ALB. It shows a tendency to be superior to the compound marker, such as GPS. Furthermore, CAR combined with NIH risk classi cation can effectively improve the prognostic prediction performance of standard NIH risk classi cation.
Extensive studies have shown that systemic in ammation plays an essential regulatory role in malignant tumors' occurrence and development [24][25][26]. In ammatory factors, including acute-phase proteins such as C-reactive protein and albumin, may come from the systemic response to malignant tumors or from the secretion of malignant cells [27]. The interaction between host and tumor cells may lead to tumor progression or retraction [27]. Therefore, several studies support the theory that the changes in systemic in ammatory factors have a particular prognostic value for cancer [8][9][10][11]. To predict the prognosis of patients with malignant tumors more accurately and facilitate clinical application, recent studies have veri ed some in ammatory indicators obtained by routine serum examination, such as CRP, albumin, GPS and CAR[28-31].
However, there are relatively few studies on these markers' prognostic signi cance in patients with GIST, let alone CAR. CAR was initially developed to identify critically ill patients in emergency medical wards and to predict 90-day mortality in patients with sepsis [32]. Recent studies have reported that CAR has potential prognostic value in hepatocellular carcinoma, gastric cancer, and other malignant tumors [33]. However, the effect of CAR on the long-term survival of patients with GIST is not apparent. Few studies have explored the prognostic in uence of preoperative CAR in GIST patients undergoing surgery. In the eld of GIST surgery, the current research established the clinical value of the preoperative CAR. Therefore, CAR is a promising prognostic marker based on in ammation in patients with GIST undergoing radical resection. It provides more prognostic information outside the existing NIH risk classi cation system, helps us to identify high-risk patients more accurately, and may help to improve treatment decision-making and prognosis.
The current research indicates that CAR is an independent prognostic factor for OS in multivariate analysis. Similar to the results of this study, previous studies have con rmed that CAR was an independent prognostic factor in patients with esophagogastric junction (AEG) and upper gastric cancer (UGC) [34]. Another retrospective study of radical resection of 455 gastric cancer cases is also consistent with our results [35].
GPS is recognized and proved to be one of the best prognostic assessments based on in ammation in different types of tumors, including GIST [36]. Therefore, it is imperative to compare the prediction ability of CAR and GPS. In this study, CRP, ALB, and GPS were also critical prognostic factors in univariate analysis. However, after using forward stepwise regression to control confounding factors, the only CAR was still a signi cant prognostic factor. In the time-dependent ROC analysis, compared with CRP, ALB, and GPS, CAR's prediction ability is better than that of CRP and ALB. It tended to be better than GPS, but the difference was not signi cant. These results are in agreement with those of Takahiro Toyokawa et al. [37]. They compared the preoperative in ammatory biomarkers in 225 patients with stage gastric carcinoma who underwent R0 resection. The results indicated CAR was an independent predictor affecting OS and cancer-speci c survival (CSS), while GPS was not. They found that the AUC of CAR and GPS was similar. Also, there was a strong association between CAR and GPS. The reason why GPS did not become an independent prognostic factor in the multivariate analysis might be that it has a strong association with CAR and its predictive ability is weaker than that of CAR. Therefore, we found that compared with the proven in ammatory marker GPS, CAR could also precisely predict the survival of patients with GIST after radical surgery. These results suggest that CAR is a new and more potential in ammation-based prognostic score in GIST.
Moreover, the CAR in the current study showed reasonable clinical practicability in patients with different clinical characteristics. When subgroups by sex, age, and ASA PS status were considered, the association between high CAR and decreased OS remained unchanged. However, this study also shows that high CAR is related to the tumor's high degree of malignancy. The tumor microenvironment of gastrointestinal stromal tumors is often characterized by apparent in ammatory cell in ltration, but the mechanism and process of the interaction between in ammation and tumor are complex. Little is known about the mechanism by which systemic in ammation regulates tumor behavior and the host state.
Studies have found that CRP increases in patients with various tumors in recent years, which may be due to the systemic response to acute or chronic in ammation. As a typical manifestation of acute response, the rapid increase of CRP is related to Cytokines that promote in ammation, such as TNF-α and IL-6[27].
Besides, DOG1 is a landmark protein of gastrointestinal stromal tumors (GIST), belonging to Ca 2+activated chloride channels and phospholipid disruptors. IL-6 can regulate cell death and tumorigenicity by regulating DOG1 and other signal transduction pathways [38]. Moreover, the tumor microenvironment of GIST contains tumor-in ltrating immune cells, such as CD3+T cells. The type and number of tumorin ltrating in ammatory cells are related to the malignant degree and prognosis of GIST. The prognosis of GIST patients with an increased number of CD3+T cells is better. The in-depth understanding of the mechanism of systemic in ammation provides an opportunity and theoretical basis for the development of effective immunotherapy GIST [39]. Therefore, CRP is more and more widely used in monitoring cancer patients' treatment and prognosis, including GIST[27].
Albumin is closely related to nutritional status, and it is also a good indicator of immune status. Malnutrition is closely related to the decline of immune function, which will weaken the body's anti-tumor immunity. Many studies have con rmed that hypoalbuminemia was correlated with poor prognosis in different tumors, including GIST[29, 40,41].
To sum up, high-risk GIST tumor cells have a high degree of malignancy, a high risk of tumor necrosis, and high tumor consumption, resulting in a high level of systemic in ammatory response characterized by increased CRP. The increase of CRP promotes tumor progression and metastasis through a variety of cytokines and signal transduction networks. At the same time, high-risk GIST will lead to severe malnutrition and even cachexia characterized by decreased serum albumin levels, weaken the immune system, and further accelerate tumor development. It explains why we observed different prognostic effects of CAR in high-risk and low-risk subgroups in subgroup analysis. Because of the high risk of recurrence in patients with high CAR, perioperative TKI treatment may reduce recurrence and prolong survival. Besides, to detect these patients' recurrence early, more frequent follow-up should be considered.
Inevitably, several limitations should be noted. First of all, this is a single-center retrospective study in which samples are included. Data are collected for a relatively long time and may be affected by selection bias. Second, although this study's sample size is medium, there are only a small number of samples in some subgroups, which may affect the results of observation. Thirdly, Current guidelines recommend that GIST patients who undergo R0 resection and have a moderate or high risk of recurrence receive adjuvant imatinib therapy. However, the situation of patients receiving TKI adjuvant therapy in this study has not been analyzed, which would inevitably lead to deviation in the process of survival analysis.
Fourthly, the GIST in this study occurs in different organs, and its prognosis is signi cantly different, which may cause bias in statistical analysis. Finally, although the X-tile application can reduce the cut-off value uctuation caused by the change of follow-up time, the optimal cut-off value of CAR may still change if the study population is different. Therefore, a universal threshold should be determined to distinguish GIST patients with a worse prognosis for further study. In the future, we will design more rigorous prospective studies to verify our preliminary results.

Conclusions
We determined that CAR is an independent prognostic factor for OS in patients with GIST after radical resection. The combination of CAR and NIH risk classi cation can effectively improve the accuracy of identifying high-risk patients with a worse prognosis. Therefore, CAR is a useful index to predict GIST's prognosis and helps optimize the choice of postoperative treatment for patients with GIST in the future.

Acknowledgment
We are deeply grateful to all the staff of the gastrointestinal department in the Beijing Hospital for their contributions and suggestions for this study.

Funding
There is no funding support for this study.

Con icts of interest/Competing interests
We do not have any potential con ict of interest in nancial or personal relationships that will be biased against our work. Commercial parties directly or indirectly related to our terms' subject matter have not received or will receive any form of bene t. The authors claim that they have no competing interests.

Availability of data and material
The original contributions proposed in the study are included in the article / supplementary materials. For further enquiries, please contact the appropriate author.
Authors' contributions GZ and XC set the concept and designed the research. XC wrote the manuscript and participated in data collation and data analysis. GZ reviewed and edited the manuscript. JC and ZL were used to investigate and record the clinical data. GZ and JC provided suggestions and useful comments for the revision of the rst draft of this article.

Ethics approval
This study involving human participants was reviewed and approved by the Beijing Hospital Ethics Committee.

Consent to participate
The patient / participant provided a written informed consent form to participate in this study.

Consent for publication (include appropriate statements)
All the authors contributed to this article and approved the submitted version for publication.