Next Generation Sequencing-based Genetic Screening in Breast Cancer Risk for Relatives of BRCA Positive Index Cases - A Subcontinent Experience

Abstract

Purpose/Objective(s): Radiotherapy is one of the mainstay treatment modalities for the cervical cancer. In Vitro evidences suggest that radiotherapy (RT) can kill various blood cells including lymphocytes. However, there is no valid evidence from human demonstrated blood cytopenia and no knowledge of individual responses associated with immune system functions. This study aimed to apply the FACS technology to investigate the immune cells subtype distribution and changes after radiotherapy in patients with cervical cancer. Materials/Methods: The human blood samples were obtained in EDTA coated tubes in accordance to ethic committee approved prospective study, where all patients signed the informed written consent. Briefly, the blood was first centrifuged with 1600 x g, 4 C and the buffy coat was stored in 90%FBS+10%DMSO gradually to-80 C. The following fluorochrome-conjugated surface antibodies were stained: CD11b, CD45, CD4, CD8a, CD19, CD56, and CD3. The cells were then fixed and permeated, followed by staining with the intracellular antibody FOXP3 and then detected by FACS machine. The percentage of immune cell subtypes at 3 time points from 6 patients (pre-RT, 2 weeks and 4 weeks) were compared using repeated analysis of variance (ANOVA) in GraphPad Prism (version 8.01). P<0.05 level was considered as statistically significant. Results: The percentage of the CD45 + lymphocytes was significantly decreased after 4 weeks of RT (mean: 85.15 vs. 56.7, P Z 0.048). The percentages of CD45 + CD19 + B cells and a total CD45 + CD3 + T cells were changed numerically with a P-value reaching significance (P Z 0.0537 and 0.0502, respectively). The percentage of CD45 + CD3-CD56 + naturally killer cells (NK), CD11b + myeloid and CD45 + CD3 + CD8 + CD56 + natural killer T cells (NKT,), CD45 + CD3 + CD4 + FOXP3 + Treg, CD8 + Cytotoxic T cell and CD4 + Helper T cells didn't show remarkable change. For individual response, 3 patients (50%) showed increased level in myeloid subtype. 5 patients (83.3%) displayed an increased percentage of lymphocytes. 2 patients (33.3%) showed increased trend of NK cells. In addition, 4 patients (66.66%) was significantly decreased in B cells percentage. All patients displayed the same T cell trend. 2 patients (33.33%) was decreased in cytotoxic T cells after RT. Interestingly, the NKT cells change to zero after RT in all patients. For T helper cells, 1 patient (16.6%) remarkedly increased. 1 patient (16.6%) was increased and others was decreased in Treg cells percentage. Conclusion: Radiation alters the lymphocyte subtypes in cervical cancer patient, with remarkable different individual responses, suggesting the differences in radio sensitivities although small sample size. The interplay between the immune system and radiotherapy immunomodulation through differential lymphocyte killing may serve a promising blood biomarker for treatment responses. Future studies with larger number of patients are needed to confirm these findings.