In patients with previously treated metastatic colorectal cancer, the average overnight urinary excretions of six modified nucleosides varied by over 100-fold according to the nucleoside considered, being highest for pseudouridine, intermediate for 1-methyladenosine, 1-methylguanosine and 4-acetylcytidine, and lowest for cytidine and adenosine. Large differences were found between patients before liver surgery or chronochemotherapy, as well as along the 24-hours scale, that partly related to sex, co-morbidities, and primary colon or rectum site.
Consistently with our findings, large between- and within-patients variations were reported for pseudouridine, 1-methyladenosine and 1-methylguanosine in patients with breast, lung, or gastric cancer. In these patients however cytidine and adenosine urinary excretions were larger, and 4-acetylcytidine was only occasionally determined [10, 11, 41].
In our study, both complete liver metastases resection and chronochemotherapy resulted in patient-specific responses in the overnight urinary excretions of the six modified nucleosides. Increasing, stable, or decreasing trends were captured in the urinary excretions of 1-methylguanosine, 1-methyladenosine, adenosine and cytidine for all the patients in St1 or St2. Consistent increases in urinary nucleosides excretions were related to both R1 metastases resections and prolonged survival or cure in the hepatectomy patients (St1), but to disease progression on chronochemotherapy (St2). These observations suggest the tight links between these four urinary nucleosides levels and cellular proliferation, this calling for further testing as early markers of efficacy of metastases surgery and chemotherapy.
The 24-hour mesors of urinary excretion levels of the modified nucleosides in St3 ranked similarly as the overnight excretions that were determined in each study. Eight-hydroxy–2-deoxyguanosine, the ninth modified nucleoside selected for St3, was not detected in 68% of the diluted urine samples. A circadian rhythm in the urinary excretion of this oxidized form of guanosine had been previously reported both in healthy adults and in patients with multiple sclerosis, but not in diabetic patients[42,43]. Moreover, elevated levels of 8-hydroxy–2-deoxyguanosine were reported in cancer patients[34,44]. Here, owing to the dilution of the samples before injection, this modified nucleoside was only identified in 32% of the urine samples and close to the limit of quantification.
Robust correlations were found between the mesor values of pseudouridine, 1-methyladenosine, 1-methylinosine, and adenosine, on the one hand, and between those of 1-methylguanosine, N2-N2-dimethylguanosine and 4-acetylcytidine on the other hand. Cytidine excretion appeared as independent from that of the other nucleosides.
Circadian and/or 12-hour rhythms characterized the urinary excretion of pseudouridine for 48.3% of the patients (highest rate) and that of 4-acetylcytidine for 34.6% of the patients (lowest rate). Individual patients displayed marked rhythmic excretion patterns for up to seven nucleosides. Interestingly, the relative circadian amplitudes of nucleosides excretions were positively associated with the duration of rest for pseudouridine, adenosine, 4-acetylcytidine and cytidine, and with the dichotomy index I<O for 1−methylguanosine (Spearman correlation, p <0.05). These results suggested that rhythmic excretion patterns of modified nucleosides could reflect a host-mediated circadian control of cancer progression. Indeed, progression-free survival and overall survival were significantly improved in metastatic colorectal cancer patients whose I<O exceeded 97.5%. This finding has been confirmed in other oncological clinical settings[45, 46].
A main limitation of our work involves the limited sample size in St1 and 2. Healthy controls would also be needed for establishing the physiological relations between host circadian biomarkers and the rhythmic turnover of nucleic acids in healthy tissues, as reflected in the urinary excretions of the modified nucleosides. A circadian rhythm was documented in healthy adults for both 8-hydroxy–2’-deoxyguanosine, with high values in the afternoon , and for pseudouridine and N2-N2-dimethylguanosine, with highest excretions occurring between 06:00 and 12:00 . Purine and pyrimidine synthesis and catabolism are rhythmically controlled by the circadian clock in mouse liver. Clock gene Bmal1 silencing ablated these metabolic rhythms. Such Bmal1 silencing could result from host circadian disruption, as shown in mice on chronic jet lag. Here, host circadian disruption was determined using 5-day time series of rest-activity monitoring. The median value of the dichotomy index I<O in our patient population was 97.9%, as compared to 97.% for our prior cohorts of metastatic colorectal cancer patients, where this parameter was shown as an independent prognostic indicator of progression-free and overall survival. I<O was here correlated with the circadian amplitude of 1-methylguanosine, and it was significantly inter-correlated with other rest-activity parameters that helped assess circadian timing system function in individual patients. The duration of rest over the 24 hours was the host parameter, that displayed the strongest links with the rhythmic excretions of modified nucleosides, suggesting that the nucleic acids turnover pattern over the 24 hours could be predominantly tied to the rest span. However, such correlations were not significant in the additional bootstrap uncertainty check, with 90th quantiles of Spearman correlation p-value > 0.1, a finding supporting the need for confirmation within a larger sample size. Interestingly, however, short sleep duration has been found to be associated with higher mortality in a large cohort of colorectal cancer survivors .
In conclusion, we identified and ranked the main modified nucleosides that are excreted into the urines of patients with colorectal cancer metastases. Large interpatient variations were identified, that could reflect tumour or host nucleic acids turnover. Intrapatient changes in urinary nucleosides excretions over the 24-hours were partly related to the circadian rhythm in rest-activity. The clinical relevance of urinary nucleosides excretions as biomarkers was further supported by the association of 1-methylguanosine excretion after R1 hepatectomy with prolonged survival, and that of early increases in modified nucleosides despite chemotherapy with disease progression and poor survival.