Blood markers of oxidative stress are strongly associated with poorer prognosis in colorectal cancer patients

Oxidative stress has been implicated in the initiation of several cancers, including colorectal cancer (CRC). Whether it also plays a role in CRC prognosis is unclear. We assessed the associations of two oxidative stress biomarkers (Diacron's reactive oxygen metabolites [d‐ROMs] and total thiol level [TTL]) with CRC prognosis. CRC patients who were diagnosed in 2003 to 2012 and recruited into a population‐based study in Germany (n = 3361) were followed for up to 6 years. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) for the associations of d‐ROMs and TTL (measured from blood samples collected shortly after CRC diagnosis) with overall survival (OS) and disease‐specific survival (DSS) were estimated using multivariable Cox regression. Particularly pronounced associations of higher d‐ROMs with lower survival were observed in stage IV patients, with patients in the highest (vs lowest) tertile having much lower OS (HR = 1.52, 95% CI = 1.14‐2.04) and DSS (HR = 1.61, 95% CI = 1.20‐2.17). For TTL, strong inverse associations of TTL with mortality were observed within all stages. In patients of all stages, those in the highest (vs lowest) quintile had substantially higher OS (HR = 0.48, 95% CI = 0.38‐0.62) and DSS (HR = 0.52, 95% CI = 0.39‐0.69). The addition of these biomarkers to models that included age, sex, tumor stage and subsite significantly improved the prediction of CRC prognosis. The observed strong associations of higher d‐ROMs and lower TTL levels with poorer prognosis even in stage IV patients suggest that oxidative stress contributes significantly to premature mortality in CRC patients and demonstrate a large potential of these biomarkers in enhancing the prediction of CRC prognosis beyond tumor stage.

included age, sex, tumor stage and subsite significantly improved the prediction of CRC prognosis. The observed strong associations of higher d-ROMs and lower TTL levels with poorer prognosis even in stage IV patients suggest that oxidative stress contributes significantly to premature mortality in CRC patients and demonstrate a large potential of these biomarkers in enhancing the prediction of CRC prognosis beyond tumor stage. CRC. 2 These factors are also associated with oxidative stress, 3 an imbalance in the formation and removal of reactive oxygen species (ROS), 4 leading to damage of several biomolecules including DNA. [5][6][7] ROS are by-products of cellular metabolism but can also be produced by immune cells (eg, phagocytic cells) during infections. Because oxidative stress could continue to cause damage to the DNA of cancer cells after tumor initiation or could affect tumor characteristics such as aggressiveness, it could play a role in CRC prognosis. However, data on the association of oxidative stress with CRC prognosis are sparse, likely due to difficulties in measuring ROS in the human body because of their short half-life. 8 Recent development of more stable methods for quantifying oxidative stress such as Diacron's reactive oxygen metabolites (d-ROMs, an indirect evaluation of oxidant status by hydroperoxides) and total thiol level (TTL, a proxy measure of antioxidant status by free thiol groups) has strongly enhanced possibilities to evaluate the association of oxidative stress with health outcomes. [9][10][11] Epidemiological studies have shown associations of blood d-ROMs levels with lung, 12 breast, 12 and colorectal cancer risk 9,12 and with cancer mortality in lung cancer patients. 10 Higher TTL were also found to be associated with lower lung and breast cancer risk 13 and mortality in the general population. 11 Whether these biomarkers also predict CRC prognosis is unclear. One small-sized study (n = 89) from Poland has investigated the associations of different oxidative stress biomarkers (8-oxo-7,8-dihydro-2-deoxyguanosine and 8-oxo-7,8-dihydroguanine) with CRC prognosis and found lower survival in patients with higher levels of these biomarkers for oxidatively damaged DNA. 14   cancer registry indicate that about half of the eligible patients in the study region were recruited. There was also an age gradient, with higher rates of recruitment of younger patients, in whom comorbidities are often lower and chemo(radio)therapy administration and CRC survival rates are usually higher compared to older patients. 15,16 What's new?
Oxidative stress has been implicated in the initiation of several cancers, including colorectal cancer (CRC). Whether oxidative stress also plays a role in CRC prognosis remains unclear, however. In this large CRC patient cohort study, higher reactive oxygen metabolites and lower total thiol levels were strongly associated with poorer prognosis. The addition of these biomarkers to models that included age, sex, tumor stage, and sub-site significantly improved the prediction of CRC prognosis. The findings suggest that oxidative stress contributes significantly to premature mortality in CRC patients and demonstrate the potential of these biomarkers in enhancing the prediction of CRC prognosis.
However, the DACHS study did not set an upper age limit, which has merits when aiming to assess the association of oxidative stress with CRC prognosis, as oxidative stress increases with age. 11 Further details of the DACHS study have been described elsewhere. 17

| Inclusion criteria
Our study population comprised all patients diagnosed in 2003-2012 who had data on either d-ROMs or TTL (n = 3730, 90%; Figure S1).
Patients with poor hemolytic, icteric or lipemic serum samples and very low d-ROMs (<50 U.Carr) or TTL (<50 μmol/L) according to the manufacturer of the assays (n = 28), those who were not operated on for CRC (n = 50), those whose blood samples were taken within 3 days of CRC surgery or within 7 days of chemo(radio)therapy initiation (n = 141) and those with no information on tumor stage and other variables of interest (n = 150) were excluded. The cut-offs for CRC surgery and chemo(radio)therapy initiation were chosen based on reasoning, as surgery and chemo(radio)therapy are likely to have an immediate effect on d-ROMs and TTL levels. A total of 3361 patients were included in the analysis.

| Data collection
At baseline (mostly during or shortly after hospital stay for CRC surgery), trained interviewers conducted personal interviews with the participants to collect information on lifestyle factors and medical history, using a standardized questionnaire. Tumor characteristics (eg, stage and subsite) and ICD-10 codes for comorbidities that were diagnosed either prior to or at the time of CRC diagnosis were abstracted from hospital records. We used the Charlson comorbidity index (CCI), 19 adapted by Deyo et al, 20 to quantify overall comorbidity as described elsewhere. 15,21 Patients were grouped into four groups, namely, CCI scores 0 (no comorbidity), 1, 2 or 3+ (severe comorbidity).
Vital status and cause of death were ascertained from population registries and public health authorities about 3, 5 and 10 years after CRC diagnosis. About 3 years after CRC diagnosis, detailed information on treatment was collected from medical records and from questionnaires sent to patients' oncologists.

| Ascertainment of oxidative stress biomarkers
At baseline, blood samples were taken from patients (median time from diagnosis to blood sampling was 63 days, interquartile range [IQR], 23-270 days), from which serum aliquots were obtained and stored at −80 C until analysis for various measures, including d-ROMs and TTL. Whenever possible, patient recruitment and sample collection was prior to or shortly after CRC surgery and prior to chemo(radio)therapy initiation. Of patients receiving chemo(radio) therapy, approximately 40% had their samples collected prior to initiation of this treatment. Median frozen time was 7.9 (IQR, 5.8-10) years.
A previous study suggests that d-ROMs and TTL have a good longterm stability under storage conditions of −80 C. 22 Samples were analyzed in two batches in the same laboratory and under similar conditions (70% in April, 2016 and 30% in June, 2016).
Standardized assays that are used to measure d-ROMs and TTL (d-ROMs and SHp assay, respectively, both from Diacron, Grosseto, Italy) were adapted to an auto-analyzer (LX20-Pro, Beckman-Coulter, Woerden, The Netherlands) at the Laboratory for Health Protection Research (Bilthoven, The Netherlands). Of samples analyzed in the first 8 days, quality assessment showed comparable mean values, with no evidence of day-to-day variability. In brief, the d-ROMs assay evaluates the total hydroperoxide concentration in Carratelli Units (U.Carr). Each U.Carr is equivalent to 0.08 mg of hydrogen peroxide per 100 mL in the sample. 23 The higher the U.Carr, the higher the level of oxidative stress. The SHp test is a spectrophotometric test which estimates the concentration of free thiol groups (eg, sulfhydryl groups) in serum in μmol/L using information on color intensity.
Higher values indicate lower levels of oxidative stress.

| Statistical analysis
The mean values of the biomarkers, especially TTL, differed by year of blood sampling. To address this, we calculated a z-score for each patient by subtracting the mean of each year of blood sampling from the value recorded for each patient and dividing by the SD of that year. 24 This standardized the data such that the mean value of each year's sample was zero and the SD was one. We assessed the distributions of z scores of d-ROMs and TTL by baseline characteristics.
ANOVA tests were used to test the mean differences, by adjusting for age and sex.
The associations of z scores of d-ROMs and TTL (in quintiles) with overall survival (OS, mortality from any cause) and disease-specific survival (DSS, mortality from CRC) were investigated using Cox proportional hazards regression. d-ROMs and TTL violated the proportional hazards assumption, as their associations with survival outcomes became weaker after 6 years of follow-up. To address this, we censored patients at 6 years of follow-up (median follow-up time), as appropriate. Time was calculated from CRC diagnosis to the respective endpoints or end of follow-up, whichever occurred first.
We also investigated the association of the TTL to d-ROMs ratio with OS and DSS. The TTL to d-ROMs ratio might reflect a balance between antioxidant and oxidant capacities and has recently been found to predict all-cause mortality more strongly than TTL or d-ROMs alone in patients with type II diabetes. 25 Two adjustment levels were applied: (a) adjustment for sex, age, tumor stage (Union for International Cancer Control, I-IV), 26  were also added to the models because of violation of the proportional hazards assumption. We also accounted for left truncation by including "delayed entry time" in the models. In sensitivity analysis, we used the Fine and Gray method 27  Subgroup analyses according to sex, age, comorbidity, smoking status, tumor stage and subsite, MSI status, chemo(radio)therapy use, time of blood sampling (tertiles of d-ROMs and TTL instead of quintiles were used because of small numbers) were performed. Here, we used backward selection to select covariates with P < .6 (defined a priori) for the models, but age, sex and stage were forced into the models. Potential variations of associations across subgroups defined by these variables were assessed with statistical tests for interaction.
Stage-specific results were additionally illustrated with adjusted survival curves. We furthermore assessed potential linear associations of the biomarkers with all-cause mortality according tumor stage, using restricted cubic spline functions. 29 Knots were placed at the 25th, 50th and 75th percentiles. Sensitivity analyses were also conducted using different knot positions (eg, at 5th, 65th and 95th percentiles). The differences in the C-indexes of the two prediction models were tested for statistical significance using the "compareC" package in R (version 3.6.0). All other analyses were conducted with the SAS software, version 9.4 (SAS Institute, Cary, North Carolina). Statistical tests were two-sided, with a significance level of 5%.

| Characteristics of the study population
A total of 3361 patients were included in the analysis ( Figure S1).

| Associations of oxidative stress biomarkers with CRC prognosis
During a follow-up period of 6 years, 1078 (32%) deaths occurred, of which 708 (66%) were due to CRC.

| Subgroup analyses by tumor stage and subsite
In stage-specific analyses, we observed significant associations between higher d-ROMs and poorer survival in stages I-II and IV but not in stage III (Table 3 and Figure 1).  (Table 3 and Figure 2), strong inverse associations of TTL with both all-cause and CRC mortality were observed within all stages, but the associations were particularly pronounced for stages I-II and IV (P interaction = .070 for OS).
Even slightly stronger associations of higher TTL to d-ROMs ratio with lower all-cause and CRC mortality were seen within all stages, with particularly pronounced associations in stage IV patients (P interaction = .013 for OS; Table 3 and Figure 3). patients ( Figure S4). A U-shaped association was observed in stage III, with a much higher mortality in patients having z scores <−1.0 or >1.0. A strong monotonic association between higher TTL and lower mortality was seen in all stages ( Figure S5). For the TTL to d-ROMs ratio, nonlinear associations with OS were observed, with a much lower survival in patients having z-scores <0 ( Figure S6). No significant interactions between the oxidative stress biomarkers and tumor subsite for the investigated survival outcomes were observed (data not shown).
3.4 | Subgroup analyses by sex, age, comorbidity, smoking status, MSI status and chemo(radio)therapy There was a significant interaction between d-ROMs and comorbidity for OS (Table S2) (Table S3) showed particularly pronounced associations of higher d-ROMs levels with lower survival among MSI-high patients, with less clear patterns of TTL levels with survival among MSI-high patients. No significant interactions between the oxidative stress biomarkers and chemo(radio)therapy use (yes/no) for the investigated survival outcomes were observed in stage II-IV or II-III patients (data not shown).

| Subgroup analyses by time of blood sampling
Comparable associations of d-ROMs and TTL with OS were seen across strata defined by time of blood sampling with respect to CRC surgery and chemo(radio)therapy initiation (Table S4). However, the associations of TTL with OS seemed weaker in recipients of chemo(radio)therapy whose blood samples were taken within 5 months of initiation of such treatment (P interaction = .001). Also, an inverse association between d-ROMs and all-cause mortality was seen in this patient group. But among patients receiving chemo(radio)therapy, there was no difference  Table 4 shows the C-indexes of models that included age, sex, tumor stage and site and those that additionally included d-ROMs and TTL.

| DISCUSSION
Oxidative stress has been implicated in the initiation of several cancers, including CRC. Whether it also plays a role in CRC prognosis is unclear. We estimated the associations of two oxidative stress biomarkers (d-ROMs and TTL) with CRC prognosis. We found particularly poorer survival in stage IV patients with higher levels of d-ROMs. We also observed strong monotonic associations between TTL and survival within all stages, and the ratio of TTL and d-ROMs was even a stronger predictor of CRC prognosis than TTL alone. The addition of d-ROMs and TTL to prognostic models that included age, sex, tumor stage and subsite, moreover, significantly improved the prediction of CRC prognosis, overall and within all stages, with improvements being particularly pronounced in stage IV patients.
Several previous studies have reported associations of oxidative stress with poorer prognosis for patients with other types of cancer.
For example, higher d-ROMs were found to be associated with poorer OS in patients with follicular lymphoma 30 and lung cancer. 10 Higher TTL was also found to be associated with higher OS in lung cancer patients. 31 Whether these biomarkers also predict CRC prognosis has gest that an imbalanced redox state might play a major role in premature mortality in CRC patients. This hypothesis is further supported by the particularly pronounced association between the TTL to d-ROMs ratio, which reflects the balance between redox control and oxidative stress, and survival. To the best of our knowledge, our study was the first to assess this association in a cancer cohort.
The finding that the addition of these biomarkers to models that included age, sex, tumor stage and subsite significantly improves the prediction of CRC in patients of all stages and within all stages suggests that these biomarkers might also provide useful information for risk stratification of CRC patients beyond tumor stage at diagnosis.
There are several possible mechanisms through which oxidative stress might be related to CRC prognosis. First, besides tumor initiation, oxidative stress might also be involved in tumor promotion and progression. For example, cellular hypoxia is common in highly proliferating cells and induces mitochondrial ROS. 32  reflect proliferative activity of the tumor. Second, oxidative stress has been implicated in the aging process, possibly through telomere erosion and cell senescence. 35 For example, we found lower TTL and weak associations of TTL with survival in older patients, suggesting that the low TTL levels in this age group might primarily reflect the aging process. Third, it is possible that the association of oxidative stress with poorer prognosis in CRC patients is modified or partly explained by comorbidity, a prognostic factor of CRC. 36 40 Given that an unhealthy lifestyle is associated with oxidative stress 11,41 and worse health outcomes (eg, risk of longterm conditions and mortality), the adoption of a healthy lifestyle (eg, regular physical activity and smoking cessation) rather than just vitamin supplementation might help to achieve a balanced redox state and thereby also enhance CRC prognosis.
Our study has limitations. First, we excluded patients lacking information on the measured biomarkers, which could have led to selection bias. However, it is worth mentioning that the excluded sample was small (n = 310, 8%) and was not different from the included sample in terms of age (mean 69.8 vs 68.3 years) but included a somewhat lower proportion of stage IV patients (13.6% vs 19%). Second, even though d-ROMs and TTL were quantified using standardized methods, their levels might be affected by surgery or chemo(radio)therapy administration. 42 We made efforts to address this by excluding patients whose blood samples were taken <4 days after CRC surgery or <8 days after chemo(radio)therapy initiation and by also adjusting for time of blood sampling in our multivariable analy- Finally, our preliminary analyses suggested that the associations of d-ROMs and TTL with CRC prognosis might be weaker for patients with >6 years of follow-up. This suggests that it might not be possible for a one-time measurement of oxidative stress to be used for predicting CRC outcomes after longer follow-up (eg, 10+ years). We addressed this by censoring patients at 6 years of follow-up.
Major strengths of our study include recruitment of patients in a population-based setting, the use of prospective design and large sample size. Also, we were able to adjust for a large number of relevant factors such as tumor stage, age, comorbidities, and time of blood draw according to CRC surgery and chemo(radio)therapy initiation (which also takes into account whether or not a patient used CRC treatments). In particular, we were able to conduct subgroup analyses according to tumor stage, age, comorbidities, MSI status and chemo(radio)therapy use, which might be useful in understanding to what extent oxidative stress impacts CRC prognosis in specific patient groups. Also, results from the stage-specific analyses and from the assessment of the predictive accuracy of prognostic models with and without the measured oxidative stress biomarkers provide evidence on the potential for these biomarkers in enhancing risk stratification of CRC patients beyond tumor stage.
In conclusion, higher d-ROMs and lower TTL levels were associ- what extent such information could also be useful to inform treatment decisions or for monitoring treatment success in CRC patients.

ACKNOWLEDGEMENT
We would like to thank Ute Handte-Daub, Ansgar Brandhorst, Petra Bächer and Piet Beekhof for their excellent technical assistance. We are particularly grateful to the study participants, as well as the interviewers who assisted in the data collection. We also gratefully appreciate the cooperation of the below-listed clinics