Background and Objectives: This study aimed to investigate the causes of continuous deep fluctuations in the absolute lymphocyte count (ALC) in an untreated patient with Chronic Lymphocytic Leukemia (CLL), who has had a favorable prognosis since the time of diagnosis. Up until now, the patient has voluntarily chosen to adopt a predominantly vegetarian and fruitarian diet, along with prolonged periods of total fasting (ranging from 4 to 39 days) each year. Materials and Methods: For this purpose, we decided to analyze the whole transcriptome profiling of peripheral blood (PB) CD19+ cells from the patient (#1) at different time-points vs. the same cells of five other untreated CLL patients who followed a varied diet. Consequently, the CLL patients were categorized as follows: the 1st group comprised patient #1 at 20 different time-points (16 time-points during nutrition and 4 time-points during fasting), whereas the 2nd group included only one time point for each of the patients (#2, #3, #4, #5, and #6) as they followed a varied diet. We performed microarray experiments using a powerful tool, the Affymetrix Human Clariom™ D Pico Assay, to generate high-fidelity biomarker signatures. Statistical analysis was employed to identify differentially expressed genes and to perform sample clustering. Results: The lymphocytosis trend in patient #1 showed recurring fluctuations since the time of diagnosis. Interestingly, we observed that approximately 4-6 weeks after the conclusion of fasting periods, the absolute lymphocyte count was reduced by about half. The gene expression profiling analysis revealed that nine genes were statistically differently expressed between the 1st group and the 2nd group. Specifically, IGLC3, RPS26, CHPT1, and PCDH9 were under expressed in the 1st group compared to the 2nd group of CLL patients. Conversely, IGHV3-43, IGKV3D-20, PLEKHA1, CYBB, and GABRB2 were over-expressed in the 1st group when compared to the 2nd group of CLL patients. Furthermore, clustering analysis validated that all the samples from patient #1 clustered together, showing clear separation from the samples of the other CLL patients. Conclusions: This study unveiled a small gene expression signature consisting of nine genes that distinguished an untreated CLL patient who followed prolonged periods of total fasting, maintaining a gradual growth trend of lymphocytosis, compared to five untreated CLL patients with a varied diet. Future investigations focusing on patient #1 could potentially shed light on the role of prolonged periodic fasting and the implication of this specific gene signature in sustaining the lymphocytosis trend and the favorable course of the disease.
Keywords: B cells; Chronic Lymphocytic Leukemia; clustering; gene expression signature; lymphocytosis; microarrays; minimum spanning tree; prolonged periodic fasting.