Multiparametric Aging Profile in Elderly Colorectal Cancer Patients: The Emerging Prognostic Role of Telomeric Repeat Containing RNA (TERRA) in Peripheral Blood Mononuclear Cells and its Involvement in Cellular Senescence

Aging is the primary risk factor for major human diseases, including cancer. In elderly patients, minimally invasive biomarkers that capture biological rather than chronological aging are critically needed to guide prognosis and clinical management. Multiparametric aging profile based on peripheral blood samples, including immune phenotype, telomere length, thymic output, circulating senescence-associated secretory phenotype (SASP) factors, and markers of denervation and sarcopenia (C-terminal Agrin Fragment, CAF, and Neural Cell Adhesion Molecule 1, NCAM1), represent a promising strategy for monitoring frail populations. Toghether to these biomarkers, telomeric repeat-containing RNA (TERRA), a class of long noncoding RNAs transcribed from chromosome ends, influences telomere dynamics and may impact cellular senescence, thus representing an interesting emerging aging biomarker; however, its functional and prognostic relevance in aging and cancer remains to be elucidated. This study explores the role of TERRA in peripheral blood mononuclear cells (PBMCs) as a potential biomarker of biological aging and clinical outcome in elderly colorectal cancer (CRC) patients, accompanied by mechanistic in vitro analyses under senescence-inducing conditions. Seventy elderly CRC patients (aged ≥ 70 years) were evaluated at the time of surgery (baseline), and for fifty of them an additional blood sample was available one year after tumor resection (follow-up). Immune profiling was performed by flow cytometry, telomere length and thymic output were assessed by qRT-PCR, SASP factors were quantified using a Luminex platform, and markers of denervation and sarcopenia were measured by ELISA. TERRA transcript emerging from chromosome arms 1q-2q-4q-10q-13q-22q (TERRAch1-2-4-10-13-22), 15q (TERRAch15), 20q (TERRAch20), and XpYp (TERRAchXY) were quantified by qRT-PCR and evaluated in relation to other aging biomarkers. All parameters were analyzed in association with clinical outcome. To investigate the modulation of TERRA expression under drug-induced senescence conditions, PBMCs from healthy donors were activated in vitro with phytohemagglutinin (PHA) and exposed to senescence-inducing chemotherapeutic agents (doxorubicin, oxaliplatin, or 5-fluorouracil). To assess the relationship between TERRA levels and DNA damage accumulation, oxidative stress experiments were performed using hydrogen peroxide. At baseline, an aged phenotype, characterized by elevated circulating SASP markers (CRP, IL-1α, IL-6, IL-8, TNF-α, CXCL1, MMP-1), increased CAF levels, and higher frequency of activated CD8⁺ T cells, was associated with worse outcomes. In contrast, higher levels of TERRAch1-2-4-10-13-22, both at baseline and follow-up, were significantly associated with better survival, while high levels of TERRAch15 expression at baseline were linked to a reduced risk of adverse events (relapse, progression, or death). Notably, TERRA levels showed positive associations with telomere length, and patients with higher TERRAch15 expression exhibited lower frequency of senescent CD8⁺ T cells and higher CD4/CD8 ratio. In vitro, chemotherapy-induced senescence in PBMCs, characterized by overexpression of CDKN1A and CDKN2A and decreased LMNB1 mRNA levels, was consistently accompanied by increased TERRA expression. Furthermore, PBMCs exhibiting higher basal TERRAch15 levels showed significantly reduced oxidative DNA damage following hydrogen peroxide exposure, as measured by γH2AX, compared with cells expressing lower TERRAch15 levels. Overall, these findings identify a minimally invasive, multifaced aging profile that is prognostic of clinical outcome in elderly CRC patients. Of interest, TERRA expression in PBMCs emerges as a novel biomarker of biological aging with significant prognostic value in this population. Moreover, in vitro data suggest a potential role of TERRA transcripts in cellular defense mechanism against aging-related