Why does circadian timing of administration matter for immune checkpoint inhibitors’ efficacy?

In 95 mNSCLC patients on second-line nivolumab, both PFS and OS were 3 to 4 times as large in those receiving the majority of nivolumab infusions before 12:54, i.e. the median value of the per-patient median timing of infusions, with respective Hazard Ratios of an earlier progression or an earlier death of 0.258 [95% CL, 0.115–0.580] and 0.174 [0.082–0.370] [11]. Predominantly morning administration of nivolumab was significantly more effective irrespective of performance status and tumor PD-L1 expression, as shown in multivariable analyses (PFS, p = 0.001; OS, p < 0.001). The larger the proportion of infusions given before 12:54, the better the PFS and OS outcomes. Thus, median OS was 34.2 months for those patients receiving at least 67% of ICIS infusion before 12:54, 12.4 months for those given at least 67% of ICI infusions after 12:54 and 15.3 months for those receiving at least 33% of ICIs before and 33% after 12:54 (p = 0.023).

Two studies investigated whether ToD of infusions influenced the efficacy of single-agent pembrolizumab as 1st or 2nd line in 180 mNSCLC patients each [17, 18]. The early ToD group comprised patients who received <20% infusions after 16:30 whilst the late ToD group involved patients who received >20% infusions after 16:30, according to the initial methodology proposed by Qian et al. [12]. As compared to the late ToD group, the early ToD groups displayed longer PFS in both studies, i.e. 19.7 vs 6.6 months, Log Rank, p = 0.056 [17], and 9.4 vs 4.9 months, p = 0.020 [18]. Median OS was also largely prolonged in the early vs late ToD group, yet not significantly so (Fig. 1). The relevance of the ToD of the initial 4 ICI infusions was shown in a study involving 197 mNSCLC [19], using 12:00 noon as a time cut-off. Their early ToD group consisted of patients receiving at least one of the initial 4 ICI infusions before noon, whilst their late ToD group involved those receiving all initial four infusions after 12:00 noon. Median PFS was 6.5 months in the early ToD group and 3.2 months in the late group (p = 0.066). Median OS were 16.1 and 7.4 months, respectively (p = 0.003). In a multivariate analysis, HR was 0.42 [0.27–0.63] (p < 0.001) for PFS and 0.54 [0.35–0.84] (p = 0.007) for OS.

The critical relevance of early ToD of infusions over the initial 4 treatment courses was confirmed in 97 mNSCLC patients receiving 1st line pembrolizumab as a single agent (N = 41) or combined with chemotherapy (N = 56) [20]. Thus OS was significantly prolonged through the administration of three or four of the 4 initial pembrolizumab infusions before 11:45 (early ToD group) as compared to one or two (late ToD). Thus, respective 2-year survival rates were 65% and 38% (p = 0.010). Multivariable analysis confirmed that the delivery of a majority of the four initial pembrolizumab-based courses in the morning was an independent predictor of a longer OS (HR = 0.28 [0.13–0.64], p = 0.003).

In a cohort of 361 patients with metastatic NSCLC, Catozzi et al. also found that early ToD infusions of pembrolizumab, nivolumab, atezolizumab, durvalumab, or avelumab significantly prolonged overall survival. They identified 11:37 as the best discriminant cut-off time. As a result, median overall survival times were 30.3 months for the patients whose majority of ICI infusions occurred before 11:37as compared to 15.9 months for those treated later in the day (p = 0.0024) [21].

All the above studies also showed that the early ToD groups received significantly more ICI infusions as compared to the late ToD groups since efficacy was best on early ToD treatment. Such an explanation is consistent with all the results where an ICI-based treatment arm proved more effective in randomized trials. Thus ICIs withdrawal is mostly related to disease progression [22]. As a result, time to treatment failure was also found to be longer following early vs late ToD administration of nivolumab, pembrolizumab or atezolizumab in 129 mNSCLC patients, with respective median times of 14 months (CI 95%, 8.9-23.4) vs 4.9 months (2.8-13.5) [22]. There were not enough events for any comparative survival assessment at the time of the report.

In 299 patients with stage IV malignant melanoma in the MEMOIR Cohort Study, Qian et al. [12] first reported that more frequent early ToD dosing of nivolumab, pembrolizumab and/or ipilimumab nearly doubled OS as compared with late ToD administrations. The early or late ToD groups involved the patients who had received less than 20% of ICIs infusions before 16:30 or 20% or more infusions after 16:30, respectively. In a propensity score-matched (PSM) analysis of 146 patients, the relative risks of earlier disease progression or death were nearly twice as low in the early vs late ToD group. Median OS was 4·8 years [3·9–not estimable] for the late ToD group, and was not reached for the early ToD group (HR = 2·04 [1·04–4·00]; p = 0·038). ICI timing was an independent prognostic factor for OS in multivariable analyses and remained unaltered using different types of ICIs, or prior corticosteroids or brain radiotherapy.

The above ToD effects were confirmed in a cohort of 121 patients with advanced melanoma [23]. The patients receiving all initial four infusions in the afternoon (after 12:00– noon) displayed worse PFS (3.3 vs 7.6 months; p = 0.009) and worse OS (5.5 vs 24.9 months; p < 0.001) compared to those given at least one of the initial four infusions in the morning. The results were confirmed using multivariable analyses.

Another retrospective study involving 73 patients with stage IV melanoma further showed that those patients receiving more than 75% of infusions of nivolumab, pembrolizumab, or nivolumab + ipilimumab in the afternoon had a shorter median survival as compared with those receiving more morning infusions (14.9 vs. 38.1 months; HR = 0.45 [0.23–0.86]; p < 0.01) [24].

A significant association between early ToD of ICI infusions and improved patient outcomes has been reported for metastatic urothelial or renal cancers in four studies.

In 92 patients receiving single-agent ICIs for metastatic urothelial cancer in Spain or Italy, Ortega et al. [25] showed that those receiving fewer than 20% ICI infusions before 16:30, had longer PFS and OS, compared to those receiving 20% or more ICI infusions after 16:30. Median PFS ranged from 11.4 to 3.6 months (HR = 2.66 [1.53–4.63] (p = 0.001) and median OS varying from 14.0 to 6.8 months (HR = 2.62 [1.48–4.63] (p = 0.001). Early ToD of infusions also improved tumor response rate (59.3% vs 16.0%). In 201 patients receiving pembrolizumab (8% of the patients), nivolumab (61%), or dual nivolumab/ipilimumab (31%) for stage IV renal cell carcinoma (RCC), Patel et al. [26] observed that the patients receiving 25% or more of their ICI infusions in the morning (before 13:00) had significantly longer OS compared to those receiving more than 25% of infusions after 13:00. Median OS were 58 and 34 months, respectively (HR = 0.51, [0.29–0.89] (p = 0.017). Early ToD was prognostic of longer survival, independently of age, sex, tumor histology, liver or brain metastases, pre-treatment LDH, and initial ICI. Similar results were found in a study involving 56 patients receiving anti-PD-1/PDL-1 as 1st or 2nd line treatment for metastatic RCC [27]. Those patients receiving 20% or more ICI infusions after 16:30 experienced worse OS (HR = 3.1 [1.27–7.36] (p = 0.01), shorter time on treatment (HR = 2.5 [1.21–4.99] (p = 0.013) and shorter time to next treatment line (HR = 1.9, [0.96–3.71] (p = 0.067) as compared to those who received less than 20% of the infusions after 16:30. In 145 patients treated with nivolumab alone, or in combination with ipilimumab as first- or second-line treatment for metastatic RCC, Dizman et al. [28] showed consistent trends towards improved response rate, prolonged time-to-treatment failure, and OS among those patients receiving less than 25% of ICI infusions after 16:30.

In 62 patients treated with nivolumab for recurrent or metastatic squamous cell carcinoma of the esophagus, significantly superior response rate, PFS, and OS resulted from the administration of the first infusion of nivolumab before 13:00 compared to its administration after 13:00 [29]. Response rate and PFS were also significantly larger in those patients who received the majority of infusions before 13:00 during the first 3 months of treatment. In contrast, no significant differences were found in response rate, PFS, and OS as a function of whether the majority of all nivolumab infusions was given before or after 13:00.

In 248 patients receiving nivolumab monotherapy for metastatic gastric cancer, Ishizuka et al. observed significant difference in response rate, PFS and OS according to whether patients received more or fewer than 70% of ICI infusions before 14:00. The respective efficacy data were: ORR, 16.9% vs 3.3%, (P = 0.01); PFS, 2.3 vs 1.6 months (HR = 0.65; P < 0.01); OS, 7.6 vs 3.9 months, (HR = 0.64; P < 0.01) [30].

In 131 patients receiving atezolizumab ± bevacizumab as first-line treatment for advanced hepatocellular carcinoma, Pascale et al. showed that the administration of the first two treatment courses after 13:00 was associated with a significant reduction of median OS as compared to the administration of at least one of the two first courses before 13:00 (11.5 months vs 18.7 months), p = 0.015). This ToD-related difference in OS outcome was supported by multivariate analysis [31].

A meta-analysis evaluated the consistency of ToD-related efficacy findings, using published data from 13 studies involving 1663 patients with advanced non-small-cell lung cancer (47%), renal cell carcinoma (24%), melanoma (20%), urothelial cancer (5%), or esophageal carcinoma (4%). The patients had received anti-programmed cell death protein 1, or anti-programmed death-ligand 1 (98%) and/or anti-cytotoxic T-lymphocyte- associated protein 4 (anti-CTLA-4) (18%). The early ToD groups had nearly twice as long OS and PFS, as compared to late ToD-treated patients. Respective overall HRs were 0.50 [95% CI, 0.42–0.58]; P < 0.00001) for OS, and 0.51 [0.42–0.61]; P < 0.00001) for PFS [32].

A single study involving 106 patients with more than six different types of solid tumours found no difference in PFS or OS between early and late ToD infusion of pembrolizumab. No role was found for cut-off points at 12:00 noon or at 15:06 (median) or at 16:30, nor for the proportion of cycles given after this cut-off point (20% or 50%). The authors ascribed this negative result to the large heterogeneity of their patient population [33].

Thus, 17 of the 18 above study reports, involving a total of 3144 metastatic cancer patients from nine countries in 4 continents, have consistently indicated improved patient outcomes following predominant early ToD administrations of ICIs (Fig. 1a, b). However, the definition of the threshold for early ToD varies from 11:37 to 16:30, whilst the definition of the proportion of late ToD infusions associated with poor ICIs efficacy ranges from 20% to 75%.. Four studies further highlighted the critical relevance of early ToD over the initial 2-months of ICI treatment. Thus, there is a clear need both for improving precision regarding the determination of optimal timing and for assessing the current evidence for the circadian mechanisms at work for cancer chronotherapy.

Comprehensive reviews have summarized the evidence of circadian time-dependencies in drug effects, i.e. chronopharmacology, and the main mechanisms that determine chronopharmacokinetics and chronopharmacodynamics [38, 44, 56–59]. Drug exposure is moderated by the circadian regulation of Absorption, Distribution, Metabolism, Elimination and Toxicities (ADMET) mechanisms at molecular, tissue and organ levels. Circadian rhythms characterize most bioactivation, detoxification and elimination processes at the transcription, protein and enzymatic levels in the liver, the chief drug-metabolizing organ, as well as in the intestine, kidney, lung, etc [60]. Recently, sex dimorphism has emerged for circadian drug responses and their molecular mechanisms, thus supporting sex-specificities in optimal circadian-based treatments, i.e. chronotherapy [61, 62].

The occurrence of circadian patterns in clock gene expressions has been shown for several but not all rodent or human cancer cell lines in synchronized cell culture studies [63–66]. Large between-tumor variations characterize both the extent of clock gene expressions and their circadian patterns in cancer tissues from rodents or humans. A metric for molecular circadian clock functionality and timing has recently been developed using Time Teller, an artificial intelligence algorithm, that proved able to model the molecular clock from a single tissue biopsy transcriptome or RNAseq [67]. Time Teller application to human oral mucosa and breast cancer omics data reveal a molecular clock disruption in nearly half of the patients’ tumors. TimeTeller has further revealed that the survival of 209 patients with primary breast cancer treated with neoadjuvant chemotherapy was significantly less when circadian molecular clocks in cancer tissue were functional, probably due to the reduced effectiveness of chemotherapy on cancer cells with functional clocks [67]. Interestingly, the expressions of common oncogenic drivers or inhibitors such as RAS, c-MYC, NOTCH and TP53 are rhythmically controlled by the circadian clock, and also interact with it, thus supporting an overarching regulatory role of the circadian clock in cancer processes and precision cancer treatments.

Other algorithms that estimate molecular circadian clock functionality and timing have been developed such as TimeSignature [68], ZeitZeiger [69], and CYCLOPS [70]. However, the key point differentiating TimeTeller from the other algorithms is that, apart from identifying timing deviations, these do not provide any other assessments of clock functionality or other quality controls on the individual timing assessments [67].

Cancer chronotherapy aims at the delivery of anticancer agents at a time (on the 24-h scale) when efficacy can be maximized and/or toxicity can be minimized. As a result, the circadian dosing time can improve or increase the extent of treatment toxicities by up to fivefold, as shown for more than 50 agents in experimental models, and 12 of them in cancer patients [62]. Most importantly, the dosing time associated with least toxicity has achieved similar or improved efficacy both in rodents and in patients [71, 72]. Clinical trials have been conducted from the evidence provided by experimental studies in nocturnal rodents, and more recently in synchronized cell cultures as well [38, 56, 65]. Overall, the clinical relevance of daily timing has been investigated for anticancer agents in human colorectal, lung, breast, pancreas, kidney, bladder, endometrium, ovary, head and neck or hematologic cancers [62]. The discovery of oxaliplatin efficacy in colorectal cancers resulted from its chronopharmacologic administration, from Phase I to large international Phase III trials [73–75]. An individual patient-level meta-analysis involving data from 345 females and 497 males with metastatic colorectal cancer revealed that males lived significantly longer on chronomodulated oxaliplatin-5-fluouracil-leucovorin rather than on conventional chemotherapy, whilst the opposite was observed for women [76]. Further experimental and clinical data support an about 6-h delay in the optimal timing of oxaliplatin, 5-fluorouracil and irinotecan, both in mice and in cancer patients [62]. Altogether, a recent systematic review involving 18 randomized, controlled studies including a total of 2547 patients concluded that most studies provided evidence of the reduction of toxicity resulting from chronomodulated chemotherapy, while efficacy was maintained or improved [77].

Oscillating molecular circadian clocks have been identified in all the cells that participate in immune system functions [78–80]. Immune cells express circadian clock genes and display 24-h mRNA and/or protein rhythms for a wide array of functional genes. As a result, circadian rhythms characterize (i) the synthesis and release of cytokines, chemokines and cytolytic factors, (ii) the daily gating of the immune response occurring through pattern recognition receptors, (iii) phagocytosis, (iv) migration to inflamed or infected tissue and immune cells trafficking, (v) cytolytic activity, and (vi) proliferative response to antigens [81]. Consequently, alterations of circadian rhythms can lead to profound disturbances in immune responses.

In experimental models, the encounter of an immune challenge given during the middle of the light span, i.e. at Zeitgeber 7 (ZT7) 7 h after light onset, when mice are resting, has been associated with an enhancement of immune responses compared to exposure in the middle of the night (ZT19) when mice are most active [41, 82, 83]. Thus, an immune stimulus caused more dendritic cells to migrate faster from the skin to the lymph nodes following its application at daytime, as compared to night-time. The number of T-cells in lymph nodes was higher during the day, and T-cell movement from blood to lymph nodes was controlled by the circadian expression of the homing molecule Intercellular Adhesion Molecule 1 (ICAM-1) [82]. Mathematical modeling revealed that the presence of dendritic cells and T-cells in the lymph node at daytime, during the rest span of mice, increased the probability of immune interactions between antigen-presenting dendritic cells and antigen-recognizing T-cells [78]. Circadian rhythms also moderated the proliferation of T-cells in the lymph nodes at a later stage of an adaptive immune response. One week after an immune stimulus consisting of OVA peptide-loaded dendritic cells, T-cells proliferated more at daytime compared to nighttime, with largest daytime expression of proteins involved in immune regulation [84]. These rhythmic patterns were suppressed in mice lacking clock gene Bmal1 expression in T-cells [85] or in those with constitutive CLOCK gene mutation [84].

The timing when the immune challenge was given also affected the later stages of the adaptative response. The B-cells from mice vaccinated in the daytime produced more antibodies at 14 days and more antigen-specific antibodies at 28 days, compared to mice vaccinated at night [86, 87]. Day-vaccinated mice also had a stronger virus-specific T-cell response at 28 days. This effect was not seen in mice lacking Bmal1 in T-cells [86, 87].

In healthy humans, circadian variations with large amplitudes characterize the circulating counts of most blood cells, including total lymphocytes and their subsets. Total lymphocytes and mononuclear cell counts reach a low point in the morning hours, i.e. between 08:00 and 10:00 then gradually increase with a daily maximum between midnight and 02:00 at night [92]. All subpopulations of interest of T(CD4⁺) and T(CD8⁺) cells including naive, central memory, effector memory, and effector T cells display distinct and significant circadian rhythms in absolute cell counts, except for effector T(CD4) cells. Most lymphocyte subset counts nearly double over the 24-h time scale. Acrophases also clump in the early night span, thus ranging from 01:31 to 02:41 for naive, central memory, and effector memory T(CD4) and T(CD8) cells. In contrast, circulating counts in both effector T(CD8) cells and natural killer cells peak in the morning or early afternoon hours [83]. Immune cell trafficking and functions are regulated in part by hormonal secretions. For instance, cortisol [93] is rhythmically secreted by the adrenal cortex in the early morning hours with reduced levels at night. Cortisol, increases the expression of CXCR4 to mediate bone marrow homing with a 3-h delay, thereby negatively regulating the counts in naive and memory T-cells [83]. Epinephrine is secreted by the adrenal medulla primarily in the afternoon hours [94]. It recruits natural killer cells and effector T cells from the marginal pool to the circulation by increasing the expression of β-adrenergic receptors and the chemokine receptor CX3CR1 and reducing the expression of adhesion molecules [83]. Adrenergic signaling also regulates the expression of vascular cell adhesion molecule-1 [95] in the bone marrow microenvironment to induce homing of leucocytes to the bone marrow and in muscles late in the activity phase [95]. Hence, trafficking to peripheral sites occurs simultaneously, thereby resulting in anti-phasic abundance in the bloodstream.

Improved treatment responses have resulted from circadian-timed delivery of interleukin-2 and interferons in laboratory rodents. In B16 melanoma-bearing mice, maximum antitumor activity was achieved following dosing at ZT4, during the early stage of the diurnal rest span of mice, for recombinant human interferon (IFN)α A/D and at ZT16 for recombinant murine IFN-γ, i.e. shortly before the middle of the nocturnal activity span [103]. The mRNA expression of IFN receptor was rhythmic in tumor cells, and peaked synchronously with the proportion of S-phase cells, thus supporting a link between the cell cycle and immunologic tumor responses potential. Highest interferon receptor expression occurred near the middle of the rest phase of mice [104].

The administration of IFN-α or IFN-γ disrupted circadian gene Per2 mRNA expression, both in the SCN and in peripheral organs, as well as the circadian rhythm in rest-activity and body temperature, following daily dosing at ZT12 but not at ZT0. The study further revealed the ability of IFNs to disrupt both the central circadian pacemaker and peripheral clocks following dosing around the beginning of the activity span (ZT12) [104]. The circadian disruption potential of IFN was further shown through the continuous administration of IFN-α to mice using a subcutaneous implanted pump. Constant rate IFN-α infusion decreased the rhythm amplitude of locomotor activity, body temperature, leukocyte counts, and plasma corticosterone levels and suppressed the oscillation in the expression of clock genes in the liver compared to repeat daily administrations at ZT2 [105]. The chronopharmacology of interleukin-2 (IL-2) was shown in tumor-bearing rats, where constant rate infusions of IL-2 induced a 37.5% mortality rate and a 25% objective tumor response rate, whereas animals receiving a “day cycle” of IL-2 had no mortality and a 100% objective response rate [106]. Clinical Phase 1 and 2 studies have shown good tolerance despite a significant increase in dose intensity with a circadian infusion schedule compared to standard or flat continuous infusion schedules of recombinant alpha-interferon-2b in melanoma or renal cell cancer patients [107, 108]. IL-2 chronotherapy also proved to be safe, moderately toxic and active in metastatic RCC patients [109].

Circadian rhythms in key mechanisms of ICI’s efficacy were recently demonstrated in mice. Circadian Pdcd-1 mRNA expression was found in tumor-associated macrophage (TAMs), whilst PD-1-positive TAMs count also displayed a significant circadian rhythm in B16/BL6 melanoma transplanted into C57BL/6J mice [88]. Pdcd-1 was further characterized as a clock-controlled gene [88]. In this mouse model, BMS-1, a small molecule inhibitor of PD-1/PD-L1, was significantly more effective following its administration at ZT18, near the middle of the activity span, in comparison with ZT6. Both circadian times, respectively correspond to the rise and decrease in PD-1 expression on TAMs. Although a two-timepoints comparison cannot identify the optimal time of administration of BMS-1, the findings suggest that the circadian expression of PD-1 on TAMs, but not in circulating monocytes, could help select the most appropriate time of day to administer PD-1/PD-L1 inhibitors. Wang et al. [89] further demonstrated that the anti-tumor immune responses were ToD-dependent. Fourteen days after the subcutaneous engraftment of a fixed melanoma cells load, tumor volumes were significantly larger in those mice engrafted ZT21 (in the mid-to-late activity span of mice) as compared to ZT9 (in the mid-to-late rest span). This difference was abrogated in mice lacking both adaptive and innate immune cells. Interestingly, T (CD8) cells, the main target cells of ICIs were significantly more numerous in the tumors at ZT9 compared to ZT21. This difference was abrogated in mice whose Bmal1 expression was specifically suppressed in dendritic cells. Thus both above reports identify a 9-h long time span, ranging from ZT9 to ZT18, within which ICIs might be most effective. Further studies are yet mandatory to identify the optimal circadian times of ICIs, and the circadian biomarkers that will help the personalization of the chronoimmunotherapy schedules.

In humans, ICIs have an elimination half-life that ranges between 14 and 27 days, and a mean clearance of about 0.36 L/day [18, 110, 111]. As a result, the steady state is usually reached after 2–4 months [112]. Additionally, it takes 32.4 weeks to reduce T-cell PD-1 occupancy by 50%, after nivolumab discontinuation [113]. These data reveal that ICIs timing might only moderate their pharmacokinetics over the initial 2–4 months of treatment, i.e. before the reach of the steady state of ICIs blood concentrations. Furthermore, the pharmacokinetics of monoclonal antibodies have peculiar aspects and are influenced by target-mediated drug disposition and drug clearance [114], which vary over time [115, 116]. Preclinical studies have shown that pembrolizumab biodistribution depends on PD-1-mediated uptake into lymphoid organs, which oscillates with a circadian rhythm. Thus, there might be circadian changes in the clearance of monoclonal antibodies from tissues with a crucial impact on T-cell antitumor function [117].

These observations call for careful investigations of ICI chronopharmacokinetics, as potential mechanisms for the dosing time dependencies in ICI efficacy over the initial treatment cycles.

In mammals, the circadian rhythm in rest activity and that in body temperature are generated by the SCN [123], thus representing biomarkers of the coordination activity of the central circadian pacemaker [123]. In humans, the dampening of the circadian amplitude in rest-activity rhythm, measured with wrist actimetry for a week, has been associated with a significant increase in both the incidence of cancer and other diseases and all causes of mortality among the 92614 participants in the UK Biobank study [124]. In cancer patients, the most clinically relevant metric of the rest-activity rhythm is the dichotomy index (I < O), a measure of the percentage of in-bed activity counts that are less than the median of out-of-bed counts. Nearly half of 436 patients with metastatic colorectal cancer patients had robust rest-activity rhythm, with an I < O of 97.5% or more, i.e. similar to healthy subjects [125], whilst 25% had damped rhythms, and a further 25% had severe circadian disruption with an I < O below 96% [126]. Low I < O values consistently predicted for both poor PFS and OS in a pooled analysis involving these 436 patients [126], and for OS in an independent meta-analysis of 6 studies in a total of 659 cancer patient [127]. Large between patients differences have been reported for the rest-activity circadian pattern in several studies Involving patients with advanced or metastatic lung or esophagus cancer or melanoma, i.e. populations similar to those where early ToD ICIs improved outcomes. The clinical relevance of rest-activity circadian disruption has also been shown especially in lung cancer patients [128–131]. Walking exercise improved sleep quality, as assessed with the Pittsburgh Sleep Index questionnaire, and the rest-activity dichotomy index I < O, only in the lower I < O group, while no effect of the intervention was found in the good I < O group or in the whole population [132]. The restoration of a near-normal rest-activity rhythm with gefitinib was associated with decreased symptom severity and improved quality of life in a pilot study involving 10 NSCLC patients [133]. In such patients, rest-activity rhythm disturbances have been frequently associated with alterations in cortisol and melatonin secretions, core body temperature, and/or circulating immune cell subset counts [134].

Disrupted circadian patterns have been identified for cortisol secretion in individual patients with advanced or metastatic breast or ovarian cancer [135]. Such disruption was further shown as an independent prognostic factor of survival in metastatic breast cancer patients [136], as well as for advanced or metastatic patients with NSCLC [137] or RCC [138], but not for those with metastatic colorectal cancer [139].

In NSCLC patients, Sephton et al. [137] observed a significant increase in early mortality among the 29 patients with flattened diurnal salivary cortisol slope compared with the 32 patients with steep slopes that suggested a near normal circadian pattern. She found that disrupted circadian cortisol profiles were associated with both low circulating total and cytotoxic T-cell lymphocyte counts and male sex. These data were further supported by the documentation of large between-patient differences in 24-h mean level, peak-trough difference and peak time of plasma cortisol in lung cancer patients [140]. In 161 patients with advanced lung cancer, diurnal salivary cortisol slope was identified as the most critical factor influencing the psychoneurological symptom cluster in multiple linear regression models after adjusting for physical performance status and number of comorbidities [141]. The diurnal slope of salivary cortisol also proved clinically relevant in 202 renal cell cancer patients [138]. Indeed, flatter cortisol slopes were significantly associated with decreased patient survival, a finding similar to that found for this endpoint in lung cancer patients [137].

In contrast, plasma melatonin 24-h patterns appeared quite similar, with a nocturnal peak occurring at 02:00 in each healthy subject, as well as in each early or late-stage lung cancer patient. The relation between the circadian patterns in salivary melatonin and cortisol was further investigated in a case control study involving 40 patients with newly-diagnosed lung cancer and 40 healthy adults [142]. The authors reported that the group of patients had lower salivary melatonin levels and higher salivary cortisol levels with flatter slopes for both hormonal concentrations The multivariate linear regression analysis indicated that the cortisol slope and fatigue score significantly predicted the sleep quality score.

Scarce studies have examined the relationship between host circadian biomarkers and immune functions in patients with lung, kidney, bladder, or esophageal cancer or melanoma. Mazzocoli et al. [140] compared circadian rhythms in circulating Natural Killer (NK), T and B lymphocyte subsets in a group of nine NSCLC patients versus a group of 11 controls. They confirmed the well-known circadian rhythms in circulating immune cell counts in healthy controls, with maxima at daytime for T(CD8), NK-cell subset (CD8dim and CD16+), Vδ2TCR, and at night for T(CD3), T(CD4), and B cells (CD20). In a group of nine stage I–IV NSCLC patients, between-patient differences characterized the 24-h patterns in circulating T(CD3), T(CD8), and B cells. The circadian rhythmicity in circulating NK-cell counts and phagocyte activity were also disrupted in five patients with stage I–II malignant melanoma, compared to 12 healthy controls [143]. The most typical alterations were discoordination between the cytotoxicity rhythms of NK-cells and phagocytes.

The downregulation of clock gene expressions in some human cancers has been associated with altered circadian patterns in the tumor microenvironment, with a possible impact on survival or immunotherapy efficacy. In human malignant melanomas, as well as in human RCCs, the mRNA expression of most clock genes was reduced, whilst displaying disrupted circadian patterns indicative of molecular clock dysfunction, compared to corresponding nonmalignant tissue [144, 145]. Those few melanoma patients whose tumor was characterized with high BMAL1 mRNA expression had prolonged survival, possibly as a result of reduced key DNA-repair enzyme expressions, increased mutational/neoantigen load, and strong intratumoral T-cell infiltration and activation [144]. Moreover, the patients with high BMAL1 tumor expression also achieved significant clinical benefits from immune checkpoint inhibitors [144, 146]. In 11 primary RCC compared to matched healthy tissues, the mRNA expression of clock genes PER2, TIMELESS, and TIPIN was downregulated, whilst that of clock-controlled gene SERPINE1 was upregulated in the tumor cells [147]. In contrast, in the microenvironment of these tumors, the mRNA expression of clock genes BMAL1, REV-ERBα, PER1, PER2, and RORA was upregulated, whilst those of CLOCK, CRY1, and CRY2 declined [148].

The overexpression of PER 2/3, CRY2, and RORα (among other clock genes) in RCC tissues was correlated with longer OS in patients with RCC [149–151]. The survival improvement was associated with the extent of tumor infiltration with T(CD4) and T(CD8) lymphocytes [151], thus highlighting the important role of the interactions between the circadian and the immune systems.

NSCLC patients with high expression of TIMELESS or low expression of RORA, PER1, PER2, or CRY2 had a significantly worse survival prognosis [152]. TIMELESS and RORA are also significantly correlated with immune checkpoint and immune infiltration levels in NSCLC [152]. Clock-related genes were reportedly repressed in human esophageal squamous cell carcinoma samples [153, 154]. In spite of these alterations, PER2 oscillations still occurred in some human esophageal cancer cell lines [154] suggesting that the response to therapy might be enhanced by linking chronotherapy to PER2 expression pattern as a circadian biomarker of interest for personalized chronotherapy.

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