Molecular characterization of the circadian clock in paediatric leukaemia patients: a prospective study protocol

In many organisms, including humans, the timing of cellular and physiological processes is regulated by an internal time generating machinery—the circadian system. In mammals, the circadian clock comprises a central pacemaker located in the suprachiasmatic nucleus in the brain, which is entrained to the geophysical time via (mostly) light signals and in turn synchronizes peripheral clocks in every cell. Cellular clocks are formed by molecular networks of interconnected and autoregulatory transcriptional-translational feedback loops, which generate 24-h rhythms in gene and protein expression and thus account for the proper timing of cellular processes [1]. Circadian oscillations are found in about 40% of all genes. Such clock-controlled genes [2, 3] are involved in many important processes including metabolism, sleep–wake rhythm, memory formation, and immunity [4]. Circadian regulation ensures for organismal adaptation of not only cellular processes, but also physiology and behaviour to the time-varying demands of the day-night cycles [5, 6]. Dysregulation of the circadian clock is associated with pathological phenotypes including sleep and mental disorders [7‐11], obesity [12], neurodegeneration [13, 14] and cancer [15, 16].

In particular, in pathologies where the treatment can cause severe side effects in the patients, like in cancer, promising studies have reported a variation in toxic effect of anti-cancer drugs depending on the time of treatment [17‐19]. On the other hand, the treatment of the above-mentioned pathologies can further disrupt the clock and contribute to some of the observed side effects of anticancer drugs.

Thus, to investigate the circadian clock in a pathological context, particularly in cancer is timely and of great medical relevance.

Acute leukaemia (AL) is the most common form of paediatric cancer and account for approximately 30% of childhood malignancies [20]. Previously published studies reported a leukaemia specific dependency on Bmal1 and Clock in a murine model of acute myeloid leukaemia (AML) [21]. Furthermore, Bmal1 and Per2 promotors were found to be hypermethylated in RPMI8402, a human T-cell lymphoblastic leukaemia cell line [22]. Even though a significant effect of chemotherapy timing has been reported in acute lymphoblastic leukaemia (ALL) therapy [23] the mechanistic role of the molecular circadian clock in ALL remains elusive.

The planned study aims at profiling the circadian clock, via characterizing the expression of circadian clock genes (BMAL1 and PER2) in healthy children, and children suffering from ALL and AML, and to correlate it with the age group, the presence of leukaemia and the administration of anti-leukaemia therapy. In particular, it enables to investigate the expression of the circadian clock in leukaemia cells at diagnosis and under therapy, and to compare it with the circadian rhythm of healthy cells in the organism of the same patient. The expression of clock genes can be measured from biological samples like blood or saliva, and RT-qPCR assays have been established in our group for assessment of circadian gene expression levels in human samples. Preclinically, we have shown that circadian rhythms are detectable also in malignant cells, though with different degrees of dysregulation, which can influence therapy response and tumour progression in colorectal carcinoma [15, 24, 25].

In the future, the knowledge acquired through this study may potentially be used to adapt leukaemia therapies to the circadian rhythm of the patient to reduce treatment toxicity and resulting side effects leading to an overall optimization of treatment efficacy.

AL is the most common malignancy in childhood amounting to approximately 30% of all paediatric malignancies [20]. Children with AL typically present symptoms associated with bone marrow insufficiency such as fever, paleness, fatigue and petechial bleeding. Further symptoms include hepatosplenomegaly, lymphadenopathy, and musculoskeletal pain. To confirm the diagnosis of AL, peripheral blood and bone marrow samples are required to conduct cytology, cytochemistry, immunophenotyping, cytogenetics and molecular genetic testing [35]. The 5-year event-free survival of paediatric patients is 80–90% [36]. Once the diagnosis is confirmed risk stratified treatment is initiated. Treatment options include conventional chemotherapy, stem-cell transplantation, as well as immunotherapy [37]. The most severe side effects include tumorlysis-syndrome, infection, bleeding as well as HPA (Hypothalamic–Pituitary–Adrenal) axis suppression [38].

To the best of our knowledge this is the first study analysing the circadian clock in children newly diagnosed with leukaemia.

Chronomodulated treatment in cancer takes advantage of the circadian rhythms in cancer and/or host cells. In fact, cancer cells have been reported to have a variety of circadian profiles, depending on the type and progression stage of a given cancer, which range from arrhythmic [39], rhythmic and in phase with healthy cells [40] or rhythmic and out of phase with healthy cells [19]. For best chronotherapy outcome, the toxicity susceptibility pattern of cancer cells should be ideally antiphase to those of healthy cells, and thus an optimal treatment timing could be set at a time point of least toxicity for healthy cells. Several animal studies [41‐43] and clinical trials with chronomodulated therapy regimen for nasopharyngeal cancer [44, 45], colorectal cancer [46], endometrial [47] and ovarian cancer [48] have shown reduced toxic side effects and improved efficacy against conventional therapy. In a recent systematic review including 18 clinical studies with 2547 patients, the authors concluded that chronomodulated chemotherapy results in the reduction of toxicity while maintaining treatment efficacy in most cases (61% of the studies) [18]. However, to our knowledge, no study has reported treatment effects on the patients’ circadian clock in healthy and leukaemia cells in ALL.

A few limitations are associated with this study including its nature as a single centre study and limited sample size, logistical challenges generated by the unforeseeable arrival of newly diagnosed patients with AL, as well as hindered collection of saliva samples, especially in very young children. Nevertheless, with this study we expect to obtain valuable inputs leading to a better understanding and potential treatment optimization of ALL, including to characterize for the first time the circadian rhythm of children with AL using sequential sampling on a systemic, as well as cellular level. To reduce bias due to the known seasonal changes in the expression of circadian clock genes [8], we aim for a continuous recruitment of subjects over the entire study-period. Furthermore, to allow for an intra-individual comparison of the clock profile between disease vs heathy cells, we will collect both leukaemic and normal cells from the same patient, at the very same time. Moreover, magnetic microbead-based cell selection has been previously established to isolate leukaemia cells, and the possibility of contamination during cell separation is negligible [49]. To quantify the specificity of the leukaemia specific markers, the percentage of healthy progenitor cells carrying this marker will be verified via the immunophenotyping included in the standard diagnostic procedure. To increase the sensitivity of the cell separation process an increased microbead concentration will be used to minimize false negatives in the flow through, as previously demonstrated [50]. Importantly, magnetic microbead-based cell selection will allow for a swift processing of newly collected samples. A deeper understanding of the mechanisms involved in potential alterations in the circadian clock of AL might contribute to a better understanding of leukaemia specific vulnerabilities and hence potentially ameliorate the efficacy of therapy and mitigate unwanted side effects and complications. Furthermore, the characterisation of the circadian clock in leukaemia cells might further contribute to a deeper understanding of clock-related mechanisms in cancer on a more general level, potentially paving the way for the implementation of chronomodulated therapy strategies or clock specific cancer therapies in a clinical setting.