The Prognostic Value of Preoperative Serum Lactate Dehydrogenase (LDH) Levels in Patients Underwent Curative-intent Hepatectomy For Colorectal Liver Metastases: A Two-Center Cohort Study

Background: The prognostic value of lactate dehydrogenase (LDH) in colorectal cancer patients has remained inconsistent between non-metastatic and metastatic settings. So far very few studies have included LDH in prognostic analysis for patients with colorectal liver metastases (CRLM) who underwent curative-intent hepatectomy. Patients and Methods: Consecutive metastatic colorectal cancer patients who underwent curative-intent resection for CRLM from two Chinese medical centers treated in 2000-2019 were enrolled in the training cohort (434 patients) and the validation cohort (146 patients). Overall survival (OS) was the primary endpoint. Cox regression model was performed to identify the prognostic values of LDH and other clinicopathology variables. A modication of the established Fong scoring system comprising LDH was developed within this Chinese population. Results: In the training cohort, preoperative LDH > upper limit of normal (ULN) was the strongest independent prognostic factor both for RFS (HR 2.11, 95% condence intervals [CIs], 1.54-2.89; P < .001) and OS (HR 2.41, 95% CI, 1.72-3.39; P < .001) in multivariate analysis. 5-year survival rates were 23.7% and 52.9% in the LDH > ULN group and LDH < ULN group, respectively. These data were also conrmed in the validation cohort and then in pooled cohort. Replacing carcinoembryonic antigen (CEA) with LDH in the Fong score contributed to an improvement in the predictive value. Conclusions: Preoperative serum LDH is a reliable and independent predictor for curative-intent CRLM resection. Composite of LDH and Fong score is a potential stratication tool for CRLM resection.


Introduction
Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related mortality worldwide (1)(2)(3). The liver is the primary life-limiting distant metastatic site for CRC (4). About a quarter of CRC patients present concurrent liver metastases, and over half will develop liver metastases through the course of diseases (5) . Surgical excision-based locoregional therapy remains the only possible curative option for colorectal liver metastases (CRLM) (6). However, only about 20% of CRLM patients are candidates for curatively intended liver resection at diagnosis (7). Whereas a growing number of curative hepatectomy has been achieved through multi-disciplinary therapy within the latest decade, most patients (50%-80%) would develop further recurrence (8, 9). The survival outcomes derived from different studies remain heterogeneous, with 5-year survival rates ranging from 25% to 60% (10)(11)(12). Thus, a better selection of patients before initiating treatment is needed to re ne the therapeutic decisions.
Recent studies have shown that apart from conventional clinicopathology variables, gene expression signatures, intratumoral immune cell in ltrations, and circulating tumor cells also have a prognostic impact on colorectal cancer (10,11,13). In particular, the serum biochemical markers, namely Gammaglutamyl transferase (GGT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), also gained momentum as prognostic indicators for mCRC (14,15).
As the key enzyme in aerobic and anaerobic glycolysis, LDH plays a pivotal role in tumor metabolism by mediating the conversion of pyruvate and lactate (16). Evidence is emerging that LDH is closely related to hypoxia, angiogenesis, in ammation, and immune status in the tumor microenvironment (TME). High serum LDH levels indicate poor prognosis among various cancer entities, and promote resistance to chemo/radio/targeted therapy (17)(18)(19). However, the prognostic value of LDH in CRC has remained inconsistent between non-metastatic and metastatic settings (20)(21)(22). Elevated circulating LDH levels were reported to be an adverse prognostic factor in unresectable CRLM patients receiving systemic therapy or hepatic arterial infusion (23)(24)(25). In contrast, this effect was not evident for the overall survival of non-metastatic CRC patients (26,27). Moreover, very few studies have included LDH in the prognostic analysis of curative-intent surgery for CRLM. Therefore, it remains to be determined whether preoperative LDH levels could predict the outcome of complete CRLM resection, in which situation patients usually achieve a no-evidence-of-disease (NED) status.
To address this issue, we performed this two-center, retrospective observational study in a cohort of 580 patients with resected CRLM. Our objectives are (a) to evaluate the prognostic impact of preoperative serum LDH levels on curative-intent surgery for CRLM; (b) to integrate LDH into the established Fong scoring system within this Chinese population to improve patient strati cation for CRLM resection.

Study population
This two-center, retrospective cohort study enrolled 580 histologically proven CRLM patients who underwent curative-intent hepatectomy in Blinded for peer review (cohort 1) and Blinded for peer review (cohort 2). Cohort 1 included 434 patients from September 2000 to December 2016, while cohort 2 included 146 patients from August 2012 to June 2019. Detailed clinical information (pre-operation and post-operation clinicopathological data, blood examination, follow-up information, etc.) was retrieved from electronic and paper-based medical records from each center. The preoperative LDH values had to be available to be eligible for inclusion.
The exclusion criteria were listed as follows: 1) Peritoneal metastasis; 2) previous history of hepatectomy; 3) R2 resection of liver metastases; 4) ablation of metastatic sites or transcatheter hepatic arterial chemoembolization (TACE) within four weeks of study entry; 5) patients in in ammatory conditions; 6) previous history of malignant tumor.
The study was approved by the ethics committee of both centers and complied with the Declaration of Helsinki.

Follow-up
Overall survival (OS) was de ned as the time from hepatic resection to death from any cause or latest follow-up. Recurrence-free survival (RFS) was measured from the date of hepatic resection to con rming recurrence or death from any reason, whichever occurred rst. Patients were followed up through outpatient clinical visits or via telephone. The follow-up starts one month after the operation and ends when tumor relapse or death was veri ed, while subjects who were lost or still alive at the date of the last contact were considered censored.

Blood sample test
Data from blood examination (blood routine tests, blood chemistry tests, and tumor marker tests) were eligible for analysis if performed within two weeks before hepatectomy. The blood examination was performed by each center's laboratory. Enrolled patients were divided into LDH-normal and LDH-high groups, using the ULN (upper limit of normal) established by each center's laboratory as the cutoff value, in anticipation of elaborating a practical clinical tool for future use. The ULN of LDH at both centers was 250 U/L. Preoperative immune/in ammation-related factors (including neutrophil, lymphocyte, monocyte, and platelet counts, LMR, LNR and LPR, and C-reaction protein) were collected. LMR, LNR and LPR were de ned as absolute lymphocyte count divided by absolute monocyte, neutrophil, and platelet count, respectively.

Modi ed Clinical Risk Score establishment and validation
The clinical risk score (CRS) was calculated according to the criteria initiated by Yuman Fong (28). Brie y, ve clinical criteria: primary lymph node-positive, the disease-free interval from the diagnosis of primary tumor < 12 months, number of CRLM > 1, maximum CRLM diameter > 5 cm, preoperative CEA levels > 200 ng/ml, were assigned one point for each and total scores were de ned as CRS.
We integrated preoperative LDH levels into the CRS model to test whether the predictive ability improved. Two models were established: (a) LDH was added to the CRS model (LDH-CRS, LDH > ULN was assigned one point); (b) preoperative CEA levels were replaced by LDH levels (modi ed CRS [mCRS], LDH > ULN was assigned one point).
The discriminatory ability of models was assessed by area under curve (AUC) in the time-dependent receiver operating characteristic (ROC) analysis. Harrell's discrimination concordance index (C-index, which is de ned as the probability that predictions and outcomes are concordant) was employed to validate the predictive ability of the models.

Statistical analysis
Patients' characteristics between different groups were compared with student's t-test, chi-square, Wilcoxon rank-sum test, or Kruskal-Wallis test as statistically appropriate. The survival curves were generated using the Kaplan-Meier method and compared with the log-rank test in terms of RFS and OS.
OS was the primary endpoint. To identify independent prognostic predictors, univariate and multivariate Cox proportional hazard regression analyses were performed. The associations between baseline clinicopathologic variables (age, gender, primary tumor location, grade of differentiation, pathology, T and N stage of primary tumor, preoperative CEA and CA19-9 levels, LDH level, number of CRLM, maximum diameter of CRLM, extrahepatic metastases, surgical margin of CRLM, preoperative chemotherapy, disease-free interval from discovery of primary tumor to liver metastases) and survival outcome were explored and quanti ed by hazard ratios (HRs) and corresponding 95% con dence intervals (CIs).
Parameters with P < 0.10 in the univariate analysis were selected and further included in the multivariate analysis, relying on the ENTER algorithm with a selected level of 0.05. In the multivariable analysis for pooled population, the cohort was obligated to be an adjustment factor to exclude the confounding factor of different a liates.
KRAS and BRAF mutation was not considered for Cox regression analysis because it was not available for all patients, especially for patients in cohort 2 (Table 1). Hence a sensitivity analysis in cases with available data of KRAS mutation status was performed in a multivariable model.
Furthermore, subgroup analyses were carried out strati ed by demographic and clinicopathologic variables, and presented by forest plots.
As for the comparison of time-dependent AUC between different models, the Wilcoxon matched-pair signed-rank test was applied. 13.6%) than patients in cohort 2 (Supplementary table 1).

LDH levels and correlations with clinical characteristics
The relationship between serum LDH and clinicopathological parameters was detailed in Table 2. In summary, serum LDH levels showed no statistical difference when strati ed by demography characteristics (age, gender), primary tumor characteristics (tumor location, pathology differentiation, T and N stage, KRAS and BRAF mutation), and metastatic site characteristics (presence of extrahepatic disease, number of CRLM, perioperative chemotherapy).
However, we observed patients with a maximum diameter of CRLM ≤ 2.5cm (the median diameter) had a higher proportion of elevated LDH levels than patients with a maximum diameter of CRLM > median (24.2% vs. 9.7%, P < .001). Patients with elevated CEA also had a greater possibility of having elevated LDH than those with normal CEA levels (18.9% vs. 10.7%, P = 0.011). A similar trend was observed for CA19-9 levels (P = 0.006). Patients with synchronous CRLM had a higher proportion of elevated LDH than those with metachronous CRLM (17.9% vs. 10.1%, P = 0.033). In addition, patients with CRS of 4-5 had higher proportion of elevated LDH than patients with CRS 2-3 or CRS 0-1 (51.2% vs. 14.2% vs. 8.7%; P < .001).
Cox regression analysis of relapse-free survival and overall survival Due to some missing data for the baseline variables (details in Table 1), 490 patients were nally included in the multivariable model. Elevated preoperative LDH levels (de ned as LDH > ULN) was found to be the strongest prognostic factor for OS (Table 3).
In the univariate analysis, age, pathology differentiation, T stage of the primary tumor, lymph node metastases of the primary tumor, preoperative CEA and CA19-9 level, number of CRLM, maximum diameter of CRLM, presence of extrahepatic metastases, preoperative chemotherapy, R0 resection margin, and LDH levels were signi cant predictors for OS.
After adjusted for the above clinicopathologic parameters, eight factors were ultimately identi ed as independent prognostic makers for OS in the multivariate analysis: age (HR, In terms of RFS, LDH remained an independent predictor in the multivariate analysis (HR, 1.53; 95% CI, 1.01-2.03; P = 0.042), along with lymph node metastases of primary tumor, number of CRLM, and maximum diameter of CRLM (supplementary Table 4).
Additionally, in the sensitivity analysis in cases with available data of KRAS mutation status, only number and size of CRLM were independent predictors for OS in multivariable models, probably due to the limitation of sample size (Supplementary Table 5 (Fig. 3).
Receiver operating characteristic (ROC) analysis for the comparison of CRS and LDH-CRS in prediction ability Time-dependent ROC analysis displayed that LDH-CRS and mCRS exhibited a better predictive value than CRS in the pooled cohort for OS (P = 0.016). In the CRS model, the C-index of the 5-year OS probability forecast was 0.653±0.029, the c-index of the LDH-CRS model was 0.674±0.029, while the c-index of the mCRS model was 0.681±0.028 (Fig. 4). These results suggest that adding LDH to the CRS scoring system demonstrated a better accuracy.

Association of LDH levels and immune/in ammation-related indexes
In an exploratory analysis, it is interesting to note that LDH levels varied with a set of immune/in ammatory factors (Fig. 5). Speci cally, patients with elevated LDH had higher preoperative neutrophil counts (P = 0.031), higher C-reaction protein (CRP) levels (P < .001), and lower lymphocyte counts (P = 0.022) than patients with normal LDH levels. Consequently, patients with elevated LDH also had a lower lymphocyte-to-monocyte ratio (LMR; P < .001) and lymphocyte-to-neutrophil ratio (LNR; P < .001). On the contrary, LDH levels were not associated with preoperative total white blood cell counts or monocyte counts.

Discussion
Resection of colorectal liver metastases is fraught with high rates of recurrence. It represents an area of intense investigation in desperate need of predictive biomarkers to aid in surgical decision-making. In the current study, we found that LDH was the strongest prognostic factor for OS both in the univariate and the multivariate analysis. Patients with elevated LDH had a nearly two-fold higher risk for mortality (mOS, 27.6 months vs. 68.8 months). The 5-year survival rate in the normal-LDH and the high-LDH groups was 53.7% and 22.5%, respectively (Fig. 1). Although some scholars have investigated the utility of LDH as a serum biomarker in resectable and unresectable CRC, its usefulness has been limited by underpowered studies as well as its non-speci city (20)(21)(22)(23)(24)(25)(26)(27). To the best of our knowledge, our study is the rst to address the independent prognostic impact of preoperative LDH levels in curative-intent CRLM resection.
Increased LDH is closely linked to hypoxia and angiogenesis in aggressive tumor phenotypes showing accelerated growth kinetics (29)(30)(31)(32)(33). Metabolism of fast-growing cancer cells is shifted toward high glucose uptake and enhanced lactate production (34). In the TME, lactate promotes proin ammatory cytokines, such as TNF-α, IL-1, IL-6, prostaglandins, and nuclear factor-κB; enhances immune-suppressive cells, such as myeloid-derived suppressor cells (MDSCs) and dendritic cells (DCs); inhibits cytolytic cells, such as natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs); recruit tumor-associated macrophages (TAMs) and promotes their conversion into immunosuppressive phenotype (35)(36)(37). Therefore, elevated LDH is a negative prognostic biomarker not only because its key role in cancer metabolism, but also because it modulates the complex interplay between the TME and the host immune system, impacting the proliferation, invasion, and migration potential of malignant tumors (38).
Interestingly, exploratory analysis unexpectedly showed that LDH levels strongly correlated with systemic in ammation indexes, namely lymphocyte-to-monocyte ratio (LMR), lymphocyte-to-neutrophil ratio (LNR), and C-reaction protein. In contrast, this correlation was not observed for CEA levels (data not shown). It has been reported that systemic in ammation leads to lymphocytopenia and increased presence of TAMs, resulting in decreased cellular immunity (39)(40)(41)(42)(43). Meanwhile, growing evidence has shown that LDH could be a marker of diminished antitumor immunity, which inversely correlated with response to immune checkpoint blockade therapy (44). Moreover, the overexpression of hypoxia-regulating factors, such as HIF-1, Foxp3, and CCL-28, might contribute to an immunosuppressive microenvironment by recruiting myeloid-derived suppressor cells (MDSCs) and TAMs (34,45). Thus, the mechanisms or pathways regulating LDH may intersect with hypoxia and antitumor immunity (16, 33, 36). LDH may serve as an alternative indicator of systemic in ammation and immunosuppression. LDH is recently emerging as an anticancer target (46, 47). Herein, we postulate that the perioperative use of nonsteroidal anti-in ammatory drugs might decrease the recurrence risk of CRLM resection (48).
It was reported that LDH could be the product of tumor necrosis due to hypoxia, which is a sign of high tumor burden (16). In the present study, serum LDH levels did not show much relevance to most clinicopathological parameters (such as primary tumor sidedness, T and N stage, KRAS status, pathology and differentiation, and disease-free interval). Though elevated LDH were indeed associated with the maximum diameter of CRLM in our analysis, nevertheless, for patients with elevated LDH, 32.2% (30/93) of them virtually had the maximum diameter of CRLM below median value (2.5 cm) ( Table 2). It was also worth noting that LDH levels were not associated with the number of CRLM. Perhaps more importantly, subgroup analysis showed that the prognostic value of LDH was independent of the number and size of CRLM. LDH also demonstrated even strong prognostic value among patients with extra-hepatic metastases or with R1 surgical margin. Besides, LDH could distinguish the survival regardless of the Fong score (Fig. 2).
The above ndings suggested that the prognostic attribute of LDH in the current study might go beyond a simple indicator of heavier tumor burden. High LDH levels might denote aggressive biology in a way that is independent of traditional molecular and clinicopathology features. LDH might be both a metabolic and an immune surveillance prognostic biomarker. not reached). We observed even more signi cant trend for CA19-9 levels ( Supplementary Fig. 2). Therefore, combing the Fong score with LDH, with a better prognostic discriminatory ability, outperformed the Fong score. Remarkably, both LDH-CRS and mCRS identi ed a relatively higher proportion of patients in the high-risk group (score 4-6) than CRS (13.2% vs. 12.0% vs. 8.5%). Thus, they could better de ne a portrait of the optimal candidate for CRLM resection with long-term survival, as well as a picture of patients in whom direct hepatectomy may be ill-advised and further neoadjuvant or adjuvant systemic therapy would be preferable.
We acknowledge that our analysis has some limitations due to its retrospective and observational nature. Some genetic parameters, including RAS, BRAF, microsatellite status, and post-relapse treatment, were not available in some datasets. It would be meaningful to combine LDH and speci c mutations and molecular features of CRC in the future. Recurrence time was not thoroughly recorded in cohort 2.
Estimating of RFS was not stringently carried out at protocol-speci ed intervals, though most physicians assessed the tumor status every 8-12 weeks. Less frequent assessment may bias in favor of a longer RFS time. Nevertheless, this factor is less likely to in uence the primary OS outcome, which could genuinely re ect the clinical bene t of hepatectomy (49). Because the determination of the optimal cutoff value of LDH was beyond the scope of this study, we used the ULN to dichotomize this continuous variable, and the two participating centers adopted the same ULN of 250 U/ml. Finally, the enrollment dates of the two cohorts differed. Prospectively de ned resectability criteria for CRLM were not established in the study protocol, the therapeutic decisions were made by a multi-disciplinary team (MDT) in each medical center. However, since surgical interventions might outline a selection process per se, this would minimize the variations in patient selection between the two cohorts.
Anyhow, the above weaknesses had to be seen through the lens of the clear strengths. The advantage of our study resided in the large sample size, the division in two independent cohorts, the long-term followup, and the heterogeneous cohort of unselected, real-world patients. We also discovered that LDH might provide additional information on tumor metabolic and immune states. The accessibility and reproducibility of the noninvasive laboratory serum LDH test support its routine use in clinical practice. We expect future studies with prospective designs to validate our ndings, and a more explicit understanding about the molecular mechanisms of LDH in governing tumor biology.

Conclusion
Our study implicates preoperative LDH level as a reliable and independent laboratory biomarker to predict the outcome of curative-intent surgery for CRLM. Integrating LDH into the established Fong scoring system can enhance the discrimination ability. Composite of LDH and Fong score is a potential strati cation tool for CRLM resection. Prospective, international studies are needed to validate these results across diverse populations.

Declarations
Ethics approval and consent to participate The authors declare that ethical approval has been acquired from the Research Ethics Committee of Sun Yat-sen University Cancer Center and Sun Yat-sen University Sixth A liated Hospital for this retrospective analysis. All necessary informed consent required by applicable law has also been acquired from relevant patients. All methods were carried out in accordance with Helsinki guidelines. No further ethical approval was required.

Consent for publication
All authors have read and approved the nal version to be published and signed the author disclosure form.

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

Competing interests
The authors declare that they have no con icts of interest related to this research         Supplementary Files