T-Cell p16^INK4A Expression Increases Post-Transplant in Patients with Multiple Myeloma

Multiple myeloma (MM) is an incurable hematologic malignancy primarily of older adults. MM treatment decisions are based partially upon chronologic age, a measure that may not reflect the patients’ physiologic age or ability to tolerate therapy. Recently, investigators have established methodology to better quantify biologic age using the molecular marker, p16^INK4a (p16). p16 expression is a marker of cellular senescence and is strongly associated with gerontogenic models. p16 rises exponentially with chronologic aging and is influenced by external factors such as physical activity, tobacco use, and solid tumor chemotherapy. Additionally, single nucleotide polymorphisms located near the p16-encoding INK4/ARF locus are linked to age-related diseases (e.g. cardiovascular disease, diabetes, glaucoma) and decreased physical function. p16 mRNA can be measured in peripheral blood T-cells thus providing a practical means of quantifying this molecular marker of age. To determine the effects of MM therapy and autologous hematopoietic stem cell transplant (AHSCT) on biologic aging, we serially measured T-cell p16 expression in MM cohorts and aged matched controls.

Methods: 23 MM patients were divided into cohorts of newly diagnosed (ND; n=11) and prior treatment (T; n=12). T-cell isolation was performed using techniques described by Liu and colleagues (Liu et al. Aging Cell 2009); p16 expression was measured using quantitative RT-PCR. 16 MM patients underwent sequential testing, with a median 42 days between each measurement. During this time, 8 patients had no intervention and 8 received immune modulatory (IMiD) therapy. Healthy age matched control T-cells were analyzed for comparison (n=17).

Results: MM patients with a median age of 62 were evaluated; 12 with early stage ISS disease, 21 with an ECOG performance status of 0-1, and 6 with high risk FISH abnormalities. 12 MM patients had prior treatment, most included AHSCT (n=11). MM treated patients had several lines of therapy; half with 1-2 lines and half with 3+ lines. Median time from AHSCT to p16 analysis was 1003 days (range 49-3630) and transplant date was unrelated to p16 expression (p=0.9577). The healthy control median age was 60 (SD 11.77) with a range of 35-82 years. T-cell yield among MM patients and healthy controls was not significantly different. Using univariate linear regression, age correlates significantly with p16 expression in healthy controls (p=0.0039) wherein p16 is estimated to increase by 1.035 fold each year [0.05 C_ts per year].

Using multivariable regression analysis, age and MM status (ND and T), were both independently associated with p16 expression. When controlling for age, patients previously treated for MM had p16 expression that was ~3.68-fold higher than healthy controls (p=0.0003). There was no significant difference in p16 expression between ND MM patients and healthy controls (fold-change=1.15, p=0.70). MM p16 levels were significantly higher in patients with prior therapy (p=0.0439) and for those who had underwent AHSCT (p=0.0129). However, p16 was unrelated to other clinical factors such as cardiovascular disease, tobacco use, radiation, stage, absolute lymphocyte count, or renal function. 8 MM patients where p16 expression was measured pre and post-IMiD treatment, a significant change in expression was not observed (median percent change: 1.16%, p=0.11); in another 8 MM patients who were untreated, p16 levels were also unchanged over time (median percent change: -0.91%, p=0.38).

Conclusions: AHSCT significantly impacts p16 expression in T-cells from MM patients. Specifically, MM patients post-AHSCT show a 3.68 fold increase in p16 expression compared to age-matched controls. Time from transplant to assessment does not impact p16 measurements, suggesting that cellular senescence of the T-cell compartment following AHSCT is persistent. MM patients exhibit known T-cell defects including variable T-cell frequency, abnormal signal transduction, and impaired cytokine secretion. Moreover, MM AHSCT patients also show impaired immune reconstitution with changes in T-cell frequency and function. To our knowledge, this is the first report describing cellular senescence post-transplant. Health status and function are affected by AHSCT; our work is important in understanding how biologic markers of age change with intensive therapy, such as AHSCT, to better predict outcomes in future studies.