Estrella Mariel Levy

Introduction: Adaptive Natural Killer cells are a specialized NK-cell subset with enhanced antibody-dependent effector functions, making them attractive candidates for cancer immunotherapy. Although circulating adaptive NK cells have demonstrated potent antitumor activity, their presence and functional status within the breast tumor microenvironment (TME) remain largely unknown. Methods: Here, we investigated the infiltration, phenotype, functional properties, and clinical relevance of NKG2C ⁺ NK cells with adaptive features (aNK) in treatment-naïve breast cancer. Using paired peripheral blood, tumor, and adjacent non-tumoral breast tissue samples, together with functional assays and transcriptomic analyses from The Cancer Genome Atlas (TCGA), we characterized tumor-infiltrating aNK cells and evaluated their association with clinical outcome. Results: aNK cells are present within breast tumors while preserving their phenotype and enhanced effector functions. Tumor-infiltrating aNK cells retained robust antibody-dependent degranulation and IFN-γ production. Complementary studies using peripheral blood-derived aNK cells further demonstrated their superior capacity to promote dendritic cell maturation, supporting their immunoregulatory potential. Moreover, we analyzed an adaptive NK-associated transcriptional signature in TCGA breast cancer samples and found that higher signature scores were associated with a longer progression-free interval and an immune microenvironment enriched in specific CD8 ⁺ T-cell populations. Discussion: These findings indicate that aNK cells are functionally resilient to the suppressive breast TME, maintaining some specialized functions and immunoregulatory properties after tumor infiltration. Their persistence and association with favorable clinical outcome provide a rationale for further investigating their potential role in antibody-based immunotherapy and as a possible target for therapeutic strategies in BC.

Tyrosine kinase inhibitors (TKIs) have transformed chronic myeloid leukemia (CML) into a manageable disease, and discontinuation has become a feasible goal for many patients. However, molecular relapse after TKI cessation remains a challenge. This study investigated factors influencing molecular relapse-free survival (MRFS) in a real-world cohort of CML patients undergoing TKI discontinuation, focusing on clinical, molecular, and immune-related variables. Traditional prognostic tools, such as the Sokal score, showed limited capacity to predict relapse risk in this context. Instead, total treatment duration and depth of molecular response emerged as critical predictors, with longer therapy and deeper remission associated with improved outcomes. A key novel finding was the prognostic relevance of cytokine profiles, particularly interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). These cytokines were strong predictors of relapse in a decision tree model that achieved high specificity and positive predictive value in an independent validation cohort. Notably, lower IL-6 and MCP-1 levels were associated with increased relapse risk, suggesting reduced immune surveillance may contribute to recurrence. Integrating cytokine signatures with molecular markers improved prognostic accuracy, supporting immune biomarkers as complements to established clinical parameters. These findings underscore the complexity of relapse mechanisms and the need for individualized risk assessment when considering TKI discontinuation.

Treatment-free remission (TFR) is a key therapeutic goal for chronic myeloid leukemia (CML) patients in deep molecular response (DMR). While predicting patient outcome remains challenging, different NK cell populations seem crucial. We conducted an immunological sub-study from the Argentina Stop Trial (AST), including 46 patients in 2019 (AST I) and 35 new patients between 2022 and 2023 (AST II). To characterize NK cell subsets in patients attempting TFR, peripheral blood mononuclear cell samples were collected before stopping treatment and phenotype and functional characteristics were assessed by flow cytometry. Non-relapsing patients from AST I exhibited NK cell subpopulations with cytomegalovirus-related memory features, high expression of cytotoxicity markers, and robust functionality. Remarkably, though clinical variables were very similar between cohorts, significant immune differences were observed. NK cell percentage and CD16 and CD57 receptor expression levels were significantly reduced in AST II (p = 0.0051; p = 0.0222; p = 0.0033, respectively), whereas NKp46, NKp44 and PD-1 expression levels were significantly increased (p = 0.0081; p < 0.0001; p < 0.0001, respectively). NK cells from AST II patients demonstrated higher overall functionality and more memory-like subpopulations, characterized mainly by the expression of CD57, NKG2C, NKp30 and NKp46 receptors among CD56dim NK cells, also with enhanced functional performance. However, in AST II, we were unable to report an association with clinical outcome. Given the enrollment time of both cohorts and that they appear to be clinically homogeneous, we consider that COVID could be impacting the immune landscape; accordingly, serum samples from AST II, but not AST I, confirmed the presence of anti-SARS-CoV-2 IgG. The influence of the COVID pandemic and the different vaccine platforms on NK cells cannot be underestimated when evaluating the role of the immune system in cancer.

Currently, therapy for early-stage human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) is based on the combination of trastuzumab and pertuzumab plus chemotherapy in a neoadjuvant regimen. The INMUNOHER study aimed to detect immunological markers in peripheral blood and their association with treatment response. Sixty-two HER2+ BC patients were recruited. Pre-treatment samples were obtained before the start of treatment, while post-treatment samples were obtained after completing therapy and before surgery and were analyzed by flow cytometry. The pathologic complete response (pCR) rate achieved was 82.3%. The expression of the NKp30, PD-1, and TIM-3 receptors was reduced in the Natural Killer (NK)-CD56dim subset of patients who did not achieve pCR. Following therapy, many changes were found in leukocytes, including alterations in T cell lymphocyte proportions. Also, the percentage of NK cells decreased, and several phenotypic changes were observed in this population. After treatment, IFN-γ production by NK cells against HER2+-cells with or without trastuzumab was significantly reduced. HER2-targeted therapy plus chemotherapy demonstrated high efficacy in most patients, reducing the statistical power for finding immunological markers. However, NK subset phenotypes correlated better with response groups, and numerous changes in the percentage of leukocytes and T and NK cells, as well as changes in the functionality of NK cells, were observed in most patients after treatment, encouraging further research into these immune populations.

Trastuzumab (TRZ) fue el primer anticuerpo monoclonal (AcM) IgG humanizado aprobado para el tratamiento del cáncer de mama (CM). Desde su aprobación en 1998, se estima que se ha administrado a más de 2,5 millones de mujeres en todo el mundo y se encuentra en la lista de medicamentos esenciales de la OMS. TRZ ha revolucionado la terapia del CM HER2+. Tanto los mecanismos no inmunológicos como los inmunomediados explican la actividad clínica de TRZ. La asociación entre el polimorfismo del FcƴRIIIa y la eficacia terapéutica de los AcMs se ha comprobado en varios modelos. Los pacientes con los genotipos F/V y V/F presentan una mejor respuesta clínica cuando son tratados con diferentes anticuerpos. Sin embargo, existen datos controvertidos sobre la asociación con el polimorfismo FcƴRIIIa y la actividad del TRZ En este trabajo evaluamos las variantes del polimorfismo FcƴRIIIa en pacientes con CM HER2+ en terapia neoadyuvante (NA) compuesta de quimioterapia (QT) basada en taxanos y platino en combinación con TRZ y pertuzumab (PER). Si bien nuestro estudio no logo identificar ninguna asociación genética de los distintos alelos del FcgRIIIa con la respuesta al tratamiento neoadyuvante de quimioterapia en combinación con TRZ, quizás debido al bajo número de pacientes que no presentaron respuesta patológica completa (RPC), estos resultados no excluyen un papel para los FcγR.

Adaptive NK cells constitute an NK cell subpopulation, which expands after human cytomegalovirus (HCMV) infection. This subpopulation has stronger production of cytokines after CD16 stimulation, longer life and persistence than conventional NK cells and are, therefore, interesting tools for cancer immunotherapy. Since there is limited information on adaptive NK cells in cancer patients, we described this population phenotypically and functionally, by flow cytometry, in the context of HER2 + breast cancer (BC) directed therapy. We assessed HCMV status in 78 patients with BC. We found that, similarly to healthy donors (HD), a high proportion of BC patients were HCMV-positive, and nearly 72% of them had an adaptive NK cell subpopulation characterized by the loss of FcεRIγ intracellular adaptor protein or the presence of NKG2C receptor. However, in BC patients, FcεRIγ− and NKG2C + NK cell populations overlapped to a lesser extent than in HD. Otherwise, no profound phenotypic differences were found between BC patients and HD. Although FcεRIγ− or NKG2C + NK cell subsets from BC patients produced more IFN-γ than their FcεRIγ + or NKG2C− NK cell counterparts, IFN-γ production increased only when NK cells simultaneously expressed FcεRIγ− and NKG2C + , whereas in HD the presence of NKG2C marker was sufficient to display greater functionality. Furthermore, in a group of patients treated with chemotherapy and Trastuzumab plus Pertuzumab, FcεRIγ-NKG2C + and FcεRIγ-NKG2C− NK cells retained greater functionality after treatment than FcεRIγ + NKG2C− NK cells. These results suggest that the presence or magnitude of adaptive NK cell subsets might serve as a key determinant for therapeutic approaches based on antibodies directed against tumor antigens.

Triple Negative Breast Cancer (TNBC) treatment is still challenging, and immunotherapy is a potential approach in this tumor subtype. Cetuximab is an IgG1 monoclonal antibody (mAb) directed against Epidermic Growth Factor Receptor (EGFR), a protein overexpressed in a subgroup of TNBC patients and associated with poor prognosis. Previously, we demonstrated in vitro that Cetuximab triggers Ab-dependent cell cytotoxicity against TNBC cells. In this study, using co-cultures including TNBC cells, and NK and Dendritic Cells (DCs) from healthy donors, we studied the effect of Cetuximab-activated NK cells on DC function. Given that we already demonstrated that TNBC has an immunosuppressive effect on NK cells, we also tested Cetuximab combination with IL-15. We determined that Cetuximab opsonization of TNBC cells increased IFN-γ and TNF-α production by NK cells co-cultured with DCs. Moreover, we showed that NK cells activated by TNBC cells opsonized with Cetuximab promoted tumor material uptake and maturation of DCs, as well as their ability to produce IL-12. Furthermore, the stimulation with IL-15 increased the activation of NK cells and the maturation of DCs. These results suggest that IL-15 may enhance the efficacy of Cetuximab in the treatment of TNBC by promoting activation of both NK cells and DCs.

Patients with non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) have shown benefit from anti-PD-1 therapies. However, not all patients experience tumor shrinkage, durable responses or prolonged survival, demonstrating the need to find response markers. In blood samples from NSCLC and RCC patients obtained before and after anti-PD-1 treatment, we studied leukocytes by complete blood cell count, lymphocyte subsets using flow cytometry and plasma concentration of nine soluble mediators, in order to find predictive biomarkers of response and to study changes produced after anti-PD-1 therapy. In baseline samples, discriminant analysis revealed a combination of four variables that helped differentiate stable disease-response (SD-R) from progressive disease (PD) patients: augmented frequency of central memory CD4⁺ T cells and leukocyte count was associated with response while increased percentage of PD-L1⁺ natural killer cells and naïve CD4⁺ T cells was associated with lack of response. After therapy, differential changes between responders and non-responders were found in leukocytes, T cells and TIM-3⁺ T cells. Patients with progressive disease showed an increase in the frequency of TIM-3 expressing CD4⁺ and CD8⁺ T cells, whereas SD-R patients showed a decrease in these subsets. Our findings indicate that a combination of immune variables from peripheral blood (PB) could be useful to distinguish response groups in NSCLC and RCC patients treated with anti-PD-1 therapy. Frequency of TIM-3⁺ T cells showed differential changes after treatment in PD vs SD-R patients, suggesting that it may be an interesting marker for monitoring progression during therapy.

The standard treatment for Triple Negative Breast Cancer (TNBC) patients is cytotoxic chemotherapy, but it is restricted since the duration of response is usually short. Blocking the PD-1/PD-L1 pathway through monoclonal antibodies (mAbs) appears to be a promising therapeutic strategy for TNBC patients. Avelumab is a human IgG1 anti-PD-L1 mAb being tested in clinical trials that may also trigger antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells as an additional antitumor activity. In the present work, we studied in vitro Avelumab-mediated ADCC against a panel of TNBC cells with different PD-L1 expression using peripheral blood mononuclear cells (PBMC) or purified NK cells from healthy donors. We determined that Avelumab significantly enhanced NK-cell mediated cytotoxicity against TNBC cells and that tumor cells expressing higher levels of PD-L1 were more sensitive to Avelumab-mediated ADCC. IFN-γ treatment upregulated PD-L1 expression in tumor cells but had a variable impact on Avelumab-mediated ADCC, which could be related to the simultaneous effect of IFN-γ on the expression of NK cell ligands. Moreover, IL-2 and IL-15 stimulation of NK cells enhanced Avelumab-triggered cytokine production and degranulation along with increased lytic activity against tumor cells. Improving the treatment of TNBC remains still a considerable challenge. This in vitro study suggests that Avelumab-mediated ADCC, independently of the blockade of the PD-1/PD-L1 pathway, could be a valuable mechanism for tumor cell elimination in TNBC. Avelumab combination with immunomodulators such as IL-15 or IL-2 could be taken into consideration to increase the therapeutic efficacy of Avelumab in TNBC.

In a previous work, we showed that CSF-470 vaccine plus BCG and GM-CSF as adjuvants resulted in a significant benefit in the distant metastasis-free survival when comparing vaccinated vs IFN-α2b-treated high-risk cutaneous melanoma patients in a phase II study. Immune monitoring demonstrated an increase in anti-tumor innate and adaptive immunity of vaccinated patients, with a striking increase in IFN-γ secreting lymphocytes specific for melanoma antigens. In an effort to dissect the first steps of the immune response elicited by CSF-470 vaccine plus adjuvants, we evaluated, in an in vitro model, leukocyte migration, cytokine production and monocyte phagocytosis of vaccine cells. Our results demonstrate that leukocytes recruitment, mostly from the innate immune system, is an early event after CSF-470 vaccine plus BCG plus GM-CSF interaction with immune cells, possibly explained by the high expression of CCL2/MCP-1 and other chemokines by vaccine cells. Early release of TNF-α and IL-1β pro-inflammatory cytokines and efficient tumor antigens phagocytosis by monocytes take place and would probably create a favorable context for antigen processing and presentation. Although the presence of the vaccine cells hampered cytokines production stimulated by BCG in a mechanism partially mediated by TGF-β and IL-10, still significant levels of TNF-α and IL-1β could be detected. Thus, BCG was required to induce local inflammation in the presence of CSF-470 vaccine cells.