No abstract

Purine analogs are among the most effective chemotherapeutic drugs for the treatment of chronic lymphocytic leukemia (CLL). However, chemoresistance and toxicity limit their clinical use. Here, we report that the DNA polymerase inhibitor aphidicolin, which displayed negligible cytotoxicity as a single agent in primary CLL cells, markedly synergizes with fludarabine and cladribine via enhanced apoptosis. Importantly, synergy was recorded regardless of CLL prognostic markers. At the molecular level, aphidicolin enhanced purine analog-induced phosphorylation of p53 and accumulation of γH2AX, consistent with increase in DNA damage. In addition, aphidicolin delayed γH2AX disappearance that arises after removal of purine analogs, suggesting that aphidicolin causes an increase in DNA damage by impeding DNA damage repair. Similarly, aphidicolin inhibited UV-induced DNA repair known to occur primarily through the nucleotide excision repair (NER) pathway. Finally, we showed that fludarabine induced nuclear import of XPA, an indispensable factor for NER, and that XPA silencing sensitized cell lines to undergo apoptosis in response to fludarabine. Together, our data indicate that aphidicolin potentiates the cytotoxicity of purine analogs by inhibiting a DNA repair pathway that involves DNA polymerases, most likely NER, and provide a rationale for manipulating it to therapeutic advantage.

Breast cancer is the most common malignancy among women worldwide. Over the last four decades, diagnostic and therapeutic procedures have improved substantially, giving patients with localized disease a better chance of cure, and those with more advanced cancer, longer periods of disease control and survival. However, understanding and managing heterogeneity in the clinical response exhibited by patients remains a challenge. For some treatments, biomarkers are available to inform therapeutic options, assess pathological response and predict clinical outcomes. Nevertheless, some measurements are not employed universally and lack sensitivity and specificity, which might be influenced by tissue-specific alterations associated with aging and lifestyle. The first part of this article summarizes available and emerging biomarkers for clinical use, such as measurements that can be made in tumor biopsies or blood samples, including so-called liquid biopsies. The second part of this article outlines underappreciated factors that could influence the interpretation of these clinical measurements and affect treatment outcomes. For example, it has been shown that both adiposity and physical activity can modify the characteristics of tumors and surrounding tissues. In addition, evidence shows that inflammaging and immunosenescence interact with treatment and clinical outcomes and could be considered prognostic and predictive factors independently. In summary, changes to blood and tissues that reflect aging and patient characteristics, including lifestyle, are not commonly considered clinically or in research, either for practical reasons or because the supporting evidence base is developing. Thus, an aim of this article is to encourage an integrative phenomic approach in oncology research and clinical management.

Methods: We examined whether immune cell profiles differ between healthy women (n = 38) and breast cancer survivors (n = 27) within 2 years of treatment, and whether any group-differences were influenced by age, cytomegalovirus infection, cardiorespiratory fitness and body composition. Using flow cytometry, CD4+ and CD8+ T cell subsets, including naïve (NA), central memory (CM) and effector cells (EM and EMRA) were identified using CD27/CD45RA. Activation was measured by HLA-DR expression. Stem cell-like memory T cells (TSCMs) were identified using CD95/CD127. B cells, including plasmablasts, memory, immature and naïve cells were identified using CD19/CD27/CD38/CD10. Effector and regulatory Natural Killer cells were identified using CD56/CD16.Results: Compared to healthy women, CD4+ CM were +Δ21% higher among survivors (p = 0.028) and CD8+ NA were −Δ25% lower (p = 0.034). Across CD4+ and CD8+ subsets, the proportion of activated (HLA-DR+) cells was +Δ31% higher among survivors: CD4+ CM (+Δ25%), CD4+ EM (+Δ32%) and CD4+ EMRA (+Δ43%), total CD8+ (+Δ30%), CD8+ EM (+Δ30%) and CD8+ EMRA (+Δ25%) (p < 0.046). The counts of immature B cells, NK cells and CD16+ NK effector cells were higher among survivors (+Δ100%, +Δ108% and +Δ143% respectively, p < 0.04). Subsequent analyses examined whether statistically significant differences in participant characteristics, influenced immunological differences between groups. Compared to healthy women, survivors were older (56 ± 6 y vs. 45 ± 11 y), had lower cardiorespiratory fitness (V̇O2max mL kg−1 min−1: 28.8 ± 5.0 vs. 36.2 ± 8.5), lower lean mass (42.3 ± 5.0 kg vs. 48.4 ± 15.8 kg), higher body fat (36.3% ± 5.3% vs. 32.7% ± 6.4%) and higher fat mass index (FMI kg/m2: 9.5 ± 2.2 vs. 8.1 ± 2.7) (all p < 0.033). Analysis of covariance revealed divergent moderating effects of age, CMV serostatus, cardiorespiratory fitness and body composition on the differences in immune cell profiles between groups, depending on the cell type examined. Moreover, across all participants, fat mass index was positively associated with the proportion of HLA-DR+ CD4+ EMRA and CD8+ EM/EMRA T cells (Pearson correlation: r > 0.305, p < 0.019). The association between fat mass index and HLA-DR+ CD8+ EMRA T cells withstood statistical adjustment for all variables, including age, CMV serostatus, lean mass and cardiorespiratory fitness, potentially implicating these cells as contributors to inflammatory/immune-dysfunction in overweight/obesity.

Introduction: T cells are important for tumour control and surveillance, but it is not well known how breast cancer treatment impacts their characteristics in blood. Methods: Healthy women (n=38, age 45 ± 11 y), breast cancer survivors (n=27, age 56 ± 6 y) and breast cancer patients (n=5, age 44 ± 9 y) took part. Survivors comprised women who had received any form of treatment for breast cancer within 1-2 years and were considered free from disease by their oncologist. Patients were recently diagnosed women scheduled to undergo neoadjuvant chemotherapy within 2 weeks. Using flow cytometry, CD4+ and CD8+ T cell subsets, including Naïve (NA), Central memory (CM) and Effector cells (EM and EMRA) were identified using CD27/CD45RA. Activation was measured by HLA-DR expression. Age, Cytomegalovirus (CMV) serostatus, cardiorespiratory fitness and body composition were assessed. Statistical analyses were conducted between healthy women and survivors. Data for patients enabled a qualitative interpretation of immune profiles at the time of diagnosis. Results: Lymphocyte, monocyte and neutrophil counts were not different between groups. However, compared to healthy women, CD4+ CM T cells were +Δ21% higher among survivors (cells/l: 417 ± 110 vs 346 ± 161, p=0.028) and CD8+ NA T cells were −Δ25% lower (cells/l: 106 ± 46 vs 142 ± 83, p=0.034). The proportion of activated (HLA-DR+) CD4+ and CD8+ T cell subsets was +Δ31% higher among survivors compared to healthy women: CD4+ CM (+Δ25%: 35 ± 16 % vs 28 ± 6 %), CD4+ EM (+Δ32%: 54 ± 17 % vs 41 ± 9 %) and CD4+ EMRA (+Δ43%: 53 ± 21 % vs 37 ± 11 %), total CD8+ (+Δ30%: 43 ± 18 % vs 33 ± 9 %), CD8+ EM (+Δ30%: 57 ± 18 % vs 44 ± 13 %) and CD8+ EMRA T cells (+Δ25%: 55 ± 21 % vs 44 ± 12 %) (p<0.046). Compared to healthy women, survivors were older (56 ± 6 y vs 45 ± 11 y), had lower cardiorespiratory fitness (VO2max mL•kg-1•min-1: 29.0 ± 5.1 vs 36.2 ± 8.5), had lower lean mass (42.3 ± 5.1 kg vs 48.4 ± 15.8 kg), had higher body fat (36.2 ± 5.4 % vs 32.7 ± 6.4 %) and higher fat mass index (FMI kg/m2: 9.5 ± 2.3 vs 8.1 ± 2.7) (all p<0.034). Among all participants, age positively correlated with activation levels of CD4+ EMRA and CD8+ EMRA T cells and FMI positively correlated with the proportion of activated CD4+ EMRA and CD8+ EM and EMRA T cells (Pearson r>0.277 p<0.036). CMV serostatus was not different between groups and did not influence results. Compared to healthy women and survivors, patients were younger (44 ± 9 y), had lower cardiorespiratory fitness (VO2max 27.3 ± 2.2 mL•kg-1•min-1), higher body fat (40 ± 5.8%) and higher FMI (11.2 ± 3.8 kg/m2). Lean mass (44.3 ± 4.4 kg) was higher than survivors but lower than healthy women. Patterns exhibited by survivors (compared to healthy women) tended to be present among patients (e.g. an accumulation of memory T cells and a decline in NA T cells). T cell activation was closer to the levels exhibited by healthy women. Conclusion: Breast cancer survivors exhibited signs of immunosenescence, including a decline in naïve T cells and accumulations of memory T cells, which exhibited higher activation levels. Among all participants, T cell activation was positively correlated with age and fat mass index, potentially contributing to systemic inflammation and a decline in immune competency. Citation Format: Ainhoa Arana Echarri, Lauren Struszczak, Mark Beresford, John P Campbell, Robert H Jones, Rachel Butler, Dylan Thompson, James E Turner. Breast cancer survivors exhibit an accumulation of CD4+ central memory T cells, a fall in CD8+ naïve T cells and higher activation of CD4+/CD8+ memory T cells in blood, which is positively correlated with age and fat mass index [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-10.

MethodsThis study examined the effects of exercise training for 8 weeks on blood immune cell characteristics among 20 breast cancer survivors (age 56 ± 6 years, Body Mass Index 25.4 ± 3.0 kg m2) within two years of treatment. Participants were randomly allocated to a partly-supervised or a remotely-supported exercise group (n = 10 each). The partly supervised group undertook 2 supervised (laboratory-based treadmill walking and cycling) and 1 unsupervised session per week (outdoor walking) progressing from 35 to 50 min and 55% to 70% V˙O2max. The remotely-supported group received weekly exercise/outdoor walking targets (progressing from 105 to 150 min per week 55% to 70% V˙O2max) via weekly telephone calls discussing data from a fitness tracker. Immune cell counts were assessed using flow cytometry: CD4+ and CD8+ T cells (Naïve, NA; Central memory, CM; and Effector cells, EM and EMRA; using CD27/CD45RA), Stem cell-like memory T cells (TSCMs; using CD95/CD127), B cells (plasmablasts, memory, immature and naïve cells using CD19/CD27/CD38/CD10) and Natural Killer cells (effector and regulatory cells, using CD56/CD16). T cell function was assessed by unstimulated HLA-DR expression or interferon gamma (IFN-γ) production with Enzyme-linked ImmunoSpot assays following stimulation with virus or tumour-associated antigens.ResultsTotal leukocyte counts, lymphocytes, monocytes and neutrophils did not change with training (p > 0.425). Most CD4+ and CD8+ T cell subtypes, including TSCMs, and B cell and NK cell subtypes did not change (p > 0.127). However, across groups combined, the CD4+ EMRA T cell count was lower after training (cells/µl: 18 ± 33 vs. 12 ± 22, p = 0.028) and these cells were less activated on a per cell basis (HLA-DR median fluorescence intensity: 463 ± 138 vs. 420 ± 77, p = 0.018). Furthermore, the partly-supervised group showed a significant decrease in the CD4+/CD8+ ratio (3.90 ± 2.98 vs. 2.54 ± 1.29, p = 0.006) and a significant increase of regulatory NK cells (cells/µl: 16 ± 8 vs. 21 ± 10, p = 0.011). T cell IFN-γ production did not change with exercise training (p > 0.515).DiscussionIn summary, most immune cell characteristics are relatively stable with 8 weeks of exercise training among breast cancer survivors. The lower counts and activation of CD4+ EMRA T cells, might reflect an anti-immunosenescence effect of exercise.