Circadian clocks coordinate physiology and behavior with the 24h solar day to provide temporal homeostasis with the external environment. The molecular clocks that drive these intrinsic rhythmic changes are based on interlocked transcription/translation feedback loops that integrate with diverse environmental and metabolic stimuli to generate internal 24h timing. In this review we highlight recent advances in our understanding of the core molecular clock and how it utilizes diverse transcriptional and post-transcriptional mechanisms to impart temporal control onto mammalian physiology. Understanding the way in which biological rhythms are generated throughout the body may provide avenues for temporally directed therapeutics to improve health and prevent disease.

Molecular Architecture of the Mammalian Circadian Clock

The immune response may be related to cancer chemotherapy in several ways. First, the patient with advanced cancer may have depressed immunological responsiveness prior to commencement of chemotherapy. Secondly, chemotherapy itself may markedly influence the immune response. Finally, chemotherapy and immunotherapy may be used together in the treatment of specific neoplasms. Before discussing these in detail, it is pertinent to review the immune response and the techniques by which it can be assessed.

Immunological Aspects of Cancer Chemotherapy

Trainees in medicine are taught to diagnose and administer treatment as needed; time-of-day is rarely considered. Yet accumulating evidence shows that ∼half of human genes and physiologic functions follow daily rhythms. Circadian medicine aims to incorporate knowledge of these rhythms to enhance diagnosis and treatment. Interest in this approach goes back at least six decades, but the path to the clinic has been marked by starts, stops, and ambiguity. How do we move the field forward to impact clinical practice? To gain insight into successful strategies, we studied the results of more than 100 human trials that evaluated time-of-administration of drugs.

Dosing Time Matters

Circadian clocks are biological timing mechanisms that generate 24-h rhythms of physiology and behavior, exemplified by cycles of sleep/wake, hormone release, and metabolism. The adaptive value of clocks is evident when internal body clocks and daily environmental cycles are mismatched, such as in the case of shift work and jet lag or even mistimed eating, all of which are associated with physiological disruption and disease. Studies with animal and human models have also unraveled an important role of functional circadian clocks in modulating cellular and organismal responses to physiological cues (ex., food intake, exercise), pathological insults (e.g. virus and parasite infections), and medical interventions (e.g. medication). With growing knowledge of the molecular and cellular mechanisms underlying circadian physiology and pathophysiology, it is becoming possible to target circadian rhythms for disease prevention and treatment. In this review, we discuss recent advances in circadian research and the potential for therapeutic applications that take patient circadian rhythms into account in treating disease.

Circadian Rhythms, Disease and Chronotherapy:

Timing chemotherapy on the basis of the body's intrinsic circadian clock-ie, chronomodulated chemotherapy-might improve efficacy and reduce treatment toxicity. This systematic review summarises the available clinical evidence on the effects of chronomodulated chemotherapy from randomised, controlled trials in adult patients with cancer, published between the date of database inception and June 1, 2021. This study complies with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and was registered on the International Prospective Register of Systematic Reviews (CRD42020177878). The protocol was published on Oct 21, 2020, before study initiation. The primary outcome measures comprised toxicity incidence, overall survival, progression-free survival, and objective response rate. Of 1455 identified abstracts, 18 studies including 2547 patients were selected. Studies were heterogeneous in study design, treatment, and population. 14 (77%) of 18 studies reported differences among groups in toxicity. 11 (61%) studies reported that chronomodulated chemotherapy resulted in a significant decrease in toxicity while maintaining anti-cancer activity. Two (11%) studies showed that chronomodulated chemotherapy reduced some toxic effects but increased others, and one (6%) study reported worse toxicity outcomes than standard chemotherapy. Three (17%) studies reported improved efficacy (survival measures, objective response rate, or time to treatment failure) of chronomodulated chemotherapy, and no studies reported a decrease in efficacy. In conclusion, most studies provide evidence of the reduction of toxicity resulting from chronomodulated chemotherapy, while efficacy is maintained. More and larger, carefully designed, randomised, controlled trials are needed to provide recommendations for clinical practice. 

Toxicity and efficacy of chronomodulated chemotherapy: a systematic review.

Chronotherapy is no longer a novel concept in cancer treatment after approximately 20 years of development. Many clinical trials have provided strong supporting evidence that chronomodulated treatment yields better results than a traditional dosage regimen. This study aimed to evaluate the adverse reactions, effect on immune functions, and therapeutic efficacy of chronomodulated infusion versus flat intermittent infusion of cisplatin (DDP) combined with intensity-modulated radiation therapy (IMRT) in patients with locoregionally advanced nasopharyngeal carcinoma (NPC). A total of 148 patients with biopsy-diagnosed untreated stage III-IVb NPC were randomly assigned to undergo two cycles of chronomodulated infusion (study group) or flat intermittent infusion (control group) of DDP (100 mg/m2 on day 1, 21 days/cycle) synchronized with radical radiotherapy. Patients in the study group received chronomodulated infusion, with peak delivery of DDP at 16:00 pm. Patients in the control group received a routine constant rate of infusion. Both groups were treated with the same radiotherapy techniques. Over a median follow-up of 20 months, the study group had better outcomes for adverse effects and immune functions compared with the control group. During the phase of concurrent chemoradiotherapy, the incidence of nausea, vomiting, and oral mucositis in the study and control groups was 66.7% and 79.5% (p   0.05). Chronochemotherapy significantly reduced the incidence of adverse reactions and enhanced the tolerance for treatment without affecting survival. It is worth mentioning that reduced destruction of immune function is a novel area of exploration in chronotherapy research.

A randomized phase II trial of induction chemotherapy followed by cisplatin chronotherapy versus constant rate delivery combined with radiotherapy

This study assessed the prognostic value of BMAL1 and Ki-67 expression in patients with nasopharyngeal carcinoma. Level of BMAL1 mRNA was assessed in tissue specimens from 36 nasopharyngeal carcinomas and 20 nasopharyngeal chronic inflammations using quantitative reverse transcriptase-polymerase chain reaction. Expression of BMAL1 and Ki-67 proteins was analyzed immunohistochemically in 90 paired nasopharyngeal carcinoma and distant normal tissues. The Kaplan-Meier curves and the Log-rank test were used to calculate prognostic significance stratified by BMAL1 and Ki67 protein expression and the COX regression model was to analyze the multivariate prognosis. BMAL1 mRNA was significantly reduced in nasopharyngeal carcinoma (4.67 ± 0.27 versus 6.64 ± 0.51 in chronic inflammation tissues, p = 0.002). Level of BMAL1 mRNA was associated with tumor distant metastasis (3.37 ± 0.66 versus 5.04 ± 0.27 compared with non-metastasis, p = 0.011). Level of BMAL1 protein was also reduced in tumor tissues and BMAL1 expression was associated with better 1-, 3- and 5-year overall survival (OS) of cancer patients (92.6%, 69.2% and 62.3% versus 59.1%, 40.9% and 0% in patients with low BMAL1 expressed tumors; p = 0.000). BMAL1 expression and age were independent prognostic factors for OS (p = 0.032). Furthermore, Ki-67 expression was high in tumor versus normal tissues and associated with poor OS of cancer patients (p = 0.035). The Pearson correlation analysis showed that there was an inverse association between BMAL1 and Ki-67 protein expression (p = 0.021). This study demonstrated that lost BMAL1 and Ki-67 overexpression were associated with poor OS of nasopharyngeal carcinoma patients.

Prognostic significance of downregulated BMAL1 and upregulated Ki-67 proteins in nasopharyngeal carcinoma.

<h3>Purpose/Objective(s)</h3> Head and neck squamous cell carcinoma (HNSCC) is associated with high immune cell infiltration, and immunotherapy is currently one of the most promising treatments for HNSCC. Dickkopf-1 (DKK1), a Wnt-signaling inhibitor and mediator of immune activation, is highly associated with tumor progression and the tumor microenvironment and, thus, disease prognosis. However, the associations between DKK1 expression, HNSCC prognosis, and tumor-infiltrating lymphocytes remain unknown. <h3>Materials/Methods</h3> We investigated these relationships in this study by exploring DKK1-expression differences in multiple tumor tissues and normal tissues, using the TIMER, Oncomine, and UALCAN databases. We searched the Kaplan–Meier database to assess correlations between DKK1 mRNA-expression levels and clinical outcomes. Subsequently, we evaluate the correlation between DKK1 expression and tumor immune-cell infiltration using the TIMER platform and TISIDB website. In addition, we assessed DKK1, CD3, and CD4 expression in 27 head and neck tumor samples and DKK1 expression in 15 normal head and neck tissue samples by immunohistochemistry. Moreover, we used the cBioPortal and UCSC Xena databases to explore aberrant DKK1 expression during tumorigenesis. <h3>Results</h3> DKK1 significantly upregulated in HNSCC tissues versus normal tissues. DKK1 expression was related to the HNSCC tumor stage, tumor grade, histology, lymph node metastasis, and a poor clinical prognosis. DKK1 expression correlated negatively with CD3+ (P < 0.0001) and CD4+ (P < 0.0001) immune cell infiltration in HNSCC tumor tissues. Moreover, DKK1 closely correlated with genetic markers of various immune cells. The prognosis of HNSCC correlated negatively with DKK1 expression in immune cells. Copy-number variation and DNA methylation may promote abnormal DKK1 upregulation in HNSCC. These results help understand the role of DKK1 in HNSCC immunotherapy and provide a rationale for clinical research on treating HNSCC by targeting DKK1. <h3>Conclusion</h3> DKK1 may therefore serve as a prognostic indicator and a biomarker for immune infiltration in HNSCC, and potentially as a central target for improving the diagnosis and immunotherapy efficacy of HNSCC. 

Dickkopf-1 is a Prognostic Biomarker of Head and Neck Squamous Cell Carcinoma that Correlates with Immune Infiltrates

Tumors are a class of diseases characterized by altered genetic information and uncontrolled growth. Sequencing technology provide researchers with a better way to explore specific tumor pathogenesis. In recent years, single-cell sequencing technology has shone in tumor research, especially in the study of liver cancer, revealing phenomena that were unexplored by previous studies. Single-cell sequencing (SCS) is a technique for sequencing the cellular genome, transcriptome, epigenome, proteomics, or metabolomics after dissociation of tissues into single cells. Compared with traditional bulk sequencing, single-cell sequencing can dissect human tumors at single-cell resolution, finely delineate different cell types, and reveal the heterogeneity of tumor cells. In view of the diverse pathological types and complex pathogenesis of hepatocellular carcinoma (HCC), the study of the heterogeneity among tumor cells can help improve its clinical diagnosis, treatment and prognostic judgment. On this basis, SCS has revolutionized our understanding of tumor heterogeneity, tumor immune microenvironment, and clonal evolution of tumor cells. This review summarizes the basic process and development of single-cell sequencing technology and its increasing role in the field of hepatocellular carcinoma.

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Papers

A randomized phase II trial of induction chemotherapy followed by cisplatin chronotherapy versus constant rate delivery combined with radiotherapy

Prognostic significance of downregulated BMAL1 and upregulated Ki-67 proteins in nasopharyngeal carcinoma.

Dickkopf-1 is a Prognostic Biomarker of Head and Neck Squamous Cell Carcinoma that Correlates with Immune Infiltrates

Advances in single-cell sequencing technology in the field of hepatocellular carcinoma