Effect of cytotoxic chemotherapy on markers of molecular age in patients with breast cancer

Abstract

Background: Senescent cells, which express p16 (INK4a) , accumulate with aging and contribute to age-related pathology. To understand whether cytotoxic agents promote molecular aging, we measured expression of p16 (INK4a) and other senescence markers in breast cancer patients treated with adjuvant chemotherapy.

Methods: Blood and clinical information were prospectively obtained from 33 women with stage I to III breast cancer at four time points: before anthracycline-based chemotherapy, immediately after anthracycline-based chemotherapy, 3 months after anthracycline-based chemotherapy, and 12 months after anthracycline-based chemotherapy. Expression of senescence markers p16 (INK4a) and ARF mRNA was determined using TaqMan quantitative reverse-transcription polymerase chain reaction in CD3(+) T lymphocytes, telomere length was determined by Southern analysis, and senescence-associated cytokines were determined by enzyme-linked immunosorbent assay. Findings were independently assessed in a cross-sectional cohort of 176 breast cancer survivors enrolled a median of 3.4 years after treatment; 39% previously received chemotherapy. All statistical tests were two-sided.

Results: In prospectively analyzed patients, expression of p16 (INK4a) and ARF increased immediately after chemotherapy and remained elevated 12 months after treatment. Median increase in log2 p16 (INK4a) was 0.81 (interquartile range = 0.28-1.62; Wilcoxon signed-rank P < .001), or a 75% absolute increase in expression, equivalent to the increase observed over 14.7 years of chronological aging. ARF expression was comparably increased (P < .001). Increased expression of p16 (INK4a) and ARF was associated with dose-dense therapy and hematological toxicity. Expression of two senescence-associated cytokines (VEGFA and MCP1) was durably increased by adjuvant chemotherapy. Telomere length was not affected by chemotherapy. In a cross-sectional cohort, prior chemotherapy exposure was independently associated with a log2-increase in p16 (INK4a) expression of 0.57 (repeated measures model, P < .001), comparable with 10.4 years of chronological aging.

Conclusions: Adjuvant chemotherapy for breast cancer is gerontogenic, inducing cellular senescence in vivo, thereby accelerating molecular aging of hematopoietic tissues.

Figure 1.

Cohort assembly. The consort diagram for LCCC0810, the adjuvant cohort (A) shows number of evaluable patients at each time point. Thirty-nine women consented to participation. Results from the baseline sample were not available for six of the 39 patients. These six could not be included in the paired analysis; thus the analytic cohort is comprised of the 33 women for whom baseline results are available. All 33 women had at least one post-treatment sample collected. Fourteen had results at all post-treatment points, 11 were missing one time point, and 8 were missing two time points. The consort diagram for LCCC0924, the survivor cohort (B) shows 34 of 210 evaluable patients were excluded because of sorting or RNA failure (see Supplementary Methods, available online). AC = doxorubicin and cyclophosphamide.

Figure 2.

Chemotherapy-induced increase in p16 ^INK4a and ARF expression in the prospective adjuvant cohort. Mean values (dots) with 95% confidence intervals (dotted lines) of log2p16 ^INK4a (A) and log2ARF (B) from pretreatment through three post-treatment time points: immediately upon count recovery after chemotherapy and 3 months and 12 months after chemotherapy. The change from pretreatment (Pre) to the average post-treatment (Post avg) value is shown for individual patients for log2p16 ^INK4a (median change = 0.81; interquartile range [IQR] = 0.28–1.62; Wilcoxon signed-rank P < .001) (C) and log2ARF (median change = 0.89; IQR = 0.29–1.27; Wilcoxon signed-rank P < .001) (D). All statistical tests were two-sided.

Figure 3.

Expression of p16 ^INK4a and ARF by age and prior chemotherapy exposure in the cross-sectional survivor cohort. Log2p16 ^INK4a (A) and log2ARF (B) at each age according to receipt of adjuvant therapy. Dots represent the average value for each patient among the three batches. Lines represent the predicted values for chemotherapy-treated (red) and non-chemotherapy-treated (blue) patients based on the results of the repeated measures models. Log2p16 ^INK4a is statistically significantly higher in chemotherapy-treated vs non-chemotherapy-treated patients (repeated measures model P < .001). Log2ARF is not statistically significantly higher in chemotherapy-treated patients vs non-chemotherapy-treated patients (linear regression model P = .10). All statistical tests were two-sided.