Background: Racotumomab-alum is an anti-idiotype vaccine targeting the NeuGcGM3 tumor-associated ganglioside. Clinical trials in advanced cancer patients have demonstrated low toxicity, high immunogenicity and clinical benefit. The goal of this study was to identify circulating biomarkers of clinical outcome. Methods: Eighteen patients with stage IIIb/IV non-small-cell lung cancer (NSCLC) were injected with racotumomab-alum as switch maintenance therapy after first line chemotherapy. Treatment was administered until severe performance status worsening or toxicity. The frequencies of innate and adaptive lymphocytes were assessed by flow cytometry. Circulat ing factors were measured using multi-analyte flow assay kits. Results: The median overall survival was 16.5 months. Twenty-seven percent of patients were classified as long-term survivors. Patients with lower baseline frequencies of CD4+Tregs and central memory (CM) CD8+T cells displayed longer survival rates. Furthermore, higher baseline frequencies of NKT cells and a high CD8+T/CD4+Treg ratio were associated with longer survival. Interestingly, patients with significantly lower levels of effector memory (EM) CD8+T cells survived longer. The levels of NKT cells and terminal effector memory (EMRA) CD8+T cells were higher in long–term survivors in comparison with short-term survivors at post-immune samples. As expected, the ratio CD8+T/CD4+Tregs showed significantly higher values during treatment in patients with clinical benefits. Regarding serum factors, pro-tumorigenic cytokines significantly increased during treatment in poor survivors. Conclusions: In advanced NSCLC patients receiving racotumomab-alum vaccine, longer survival is associated with a unique profile of circulating lymphocyte subsets at baseline and during treatment. Additionally, certain protumor-related cytokines increased in short-term survivors. These results should be confirmed in larger clinical trials. This clinical trial was registered in Cuban Clinical Trials Register (RPCE00000279).

Background: Racotumomab-alum is an anti-idiotype vaccine targeting the NeuGcGM3 tumor-associated ganglioside. Clinical trials in advanced cancer patients have demonstrated low toxicity, high immunogenicity and clinical benefit. The goal of this study was to identify circulating biomarkers of clinical outcome. Methods: Eighteen patients with stage IIIb/IV non-small-cell lung cancer (NSCLC) were injected with racotumomab-alum as switch maintenance therapy after first-line chemotherapy. Treatment was administered until severe performance status worsening or toxicity. The frequencies of innate and adaptive lymphocytes were assessed by flow cytometry. Circulating factors were measured using multi-analyte flow assay kits. Results: The median overall survival was 16.5 months. Twenty-seven percent of patients were classified as long-term survivors. Patients with lower baseline frequencies of CD4+Tregs and central memory (CM) CD8+T cells displayed longer survival rates. Furthermore, higher baseline frequencies of NKT cells and a high CD8+T/CD4+Treg ratio were associated with longer survival. Interestingly, patients with significantly lower levels of effector memory (EM) CD8+T cells survived longer. The levels of NKT cells and terminal effector memory (EMRA) CD8+T cells were higher in long-term survivors in comparison with short-term survivors in post-immune samples. As expected, the ratio of CD8+T/CD4+Tregs showed significantly higher values during treatment in patients with clinical benefits. Regarding serum factors, pro-tumorigenic cytokines significantly increased during treatment in poor survivors. Conclusions: In advanced NSCLC patients receiving racotumomab-alum vaccine, longer survival could be associated with a unique profile of circulating lymphocyte subsets at baseline and during treatment. Additionally, certain pro-tumor-related cytokines increased in short-term survivors. These results should be confirmed in larger randomized clinical trials. This clinical trial was registered in the Cuban Clinical Trials Register (RPCE00000279).

The thymus is a primary lymphoid organ which contains stromal cell components that provide the necessary support for thymocyte development and maturation. Thymus stromal cells include, among others, thymic epithelial cells (TEC), endothelial cells, mesenchymal/fibroblast cells, and hematopoietic populations such as dendritic cells, B cells, and macrophages. TECs are particularly critical through all the stages of T cell development, contributing to the recruitment of hematopoietic precursors from the bone marrow, directing lineage specification, supporting proliferative expansion, and orchestrating both positive and negative selection processes (Cepeda & Griffith, 2018) (Han & Zúñiga-Pflücker, 2021). Anatomically, the thymus is organized into two distinct regions: the cortex in the outer zone, which contains the cortical TECs (cTECs) and the medulla in the inner zone, containing the medullary TECs (mTECs). cTECs are primarily involved in the positive selection of thymocytes. In contrast, mTECs participate in the negative selection, promoting central tolerance and supporting the development of Foxp3⁺ regulatory T cells and natural killer T cells (Han & Zúñiga-Pflücker, 2021) (Nitta & Suzuki, 2016). A young and fully functional thymus is essential for generating a pool of diverse naïve T cells, which supports robust immune responses. However, during aging, the thymus undergoes progressive involution characterized by structural and molecular modifications. These age-associated changes lead to a marked decline in the production of newly generated naïve T cells. In compensation, a homeostatic expansion of existing T cell populations occurs, resulting in reduced diversity within the peripheral T cell repertoire. Functionally, this adaptation contributes to diminished responsiveness to pathogens, increased susceptibility to autoimmune conditions, and impaired tumor surveillance (Liang et al., 2022) (Han & Zúñiga-Pflücker, 2021). The study of the thymic microenvironment and its influence on thymocyte outcome during aging provides valuable insights into the structural and functional changes that occur with senescence. One of the hallmark features of thymic aging is the decline in TEC cellularity, affecting both cTECs and mTECs subsets. TECs can be phenotypically identified with the pan-epithelial marker CD326 (EpCAM) within the CD45- thymic cell population. Further discrimination between TEC subsets could be achieved using the CD205 marker. cTECs subset could be characterized as CD45-CD326+CD205+, whereas CD45-CD326+CD205- cells correspond to mTECs (Jain & Gray, 2014). Aging is also associated with a reduction in MHC class II expression on TECs, reflecting a decline in TEC antigen-presenting ability (Gray et al., 2006). Within the TEC population, two subgroups, MHCIIlow and MHCIIhigh, can be distinguished based on the expression levels of MHC class II molecules (Jain & Gray, 2014) (Xing & Hogquist, 2014). A key molecular regulator of TEC function is the transcription factor FoxN1, which is indispensable for thymus organogenesis and TEC maintenance in adults. FoxN1 expression declines progressively with age, contributing to thymic involution and reduced naïve T cell output (Rode et al., 2015). Overexpression of FoxN1 has been shown to mitigate age-related thymic changes, restoring thymic mass, architecture, and the naïve T cell population in the spleen (Zook et al., 2011). Thymic medulla, besides containing mTECs, is populated by hematopoietic antigen presenting cells (APCs) including dendritic cells, B cells, and macrophages. Through expression and presentation of self-antigens, these APCs contribute to the process of shaping the normal T cell repertoire by negatively selecting thymocytes recognizing self-antigens. Senescence studies have demonstrated age-associated declines in APCs function and cell counts (Ki et al., 2014) (Cepeda & Griffith, 2018) (Wedemeyer & Griffith, 2025) (Van Dommelen et al., 2010). Thymocytes development starts from the double negative (DN) to simple positive (SP) T cells. At first, T cells are CD4-CD8- (DN), in the second stage they are double positive CD4+CD8+ (DP) and finally CD4+CD8- or CD4-CD8+ (SP) in the third stage. After positive selection, SP CD4+ or CD8+ T cells migrate to the thymus medulla to go through negative selection mediated by medullar TECs and APCs, where autoreactive SP T cells are deleted by apoptosis. This process of thymopoiesis results in population of peripheral blood and lymphoid sites with naive CD4+ and CD8+T cells and CD4+ regulatory T cells (Tregs) (Liang et al., 2022). Early T-lineage progenitors (ETPs) frequency declines with aging, and their potential ability to reconstitute the thymus is also reduced (Min et al., 2005). Both DN and DP population cell numbers are significantly modified with aging. DN subset frequency increases in aged mice compared with young, whereas the percentage of DP (CD4+CD8+) subpopulations significantly diminishes with age. Additionally, thymic aging is concomitant with the abnormal accumulation of CD3+ DN cells within the thymus (Aw et al., 2010). Aging also interferes with later stages of thymocyte development. DP and SP thymocytes in aged mice display deregulated CD3 expression, which may lead to attenuated TCR-dependent stimulation (Aw et al., 2010). Consistent with impaired thymocyte differentiation in the aged thymus, T-cell receptor excision circles (TRECs) within the thymus also significantly decline with aging in mice and humans (Ortman et al., 2002). Regarding thymocytes, the member of the high mobility group (HMG)-Box transcription factors TOX-1 has been identified as a mediator of thymic T cell expansion and the development of lymphoid inducer (Scott et al., 2019). The pattern of TOX expression in the thymus is highly regulated during the positive selection process. Downregulation of TOX-1 is observed in a later stage of...