Emanuela Marcenaro

Despite growing enthusiasm for immunotherapy in cancer treatment, only a few colorectal cancer (CRC) patients experience lasting therapeutic effect. This limited efficacy is likely driven by different tumor-intrinsic resistance mechanisms, including loss or downregulation of HLA-I molecules, which may impair antigen presentation and cytotoxic T cell recognition. Interestingly, the downregulation of HLA-I can augment tumor susceptibility to natural killer (NK) cell attack. However, NK cell activity may be inhibited by immunosuppressive signals within the tumor microenvironment. This study describes two patients with metastatic CRC and microsatellite instability who showed durable responses to pembrolizumab despite unfavorable prognostic factors (tumor recurrence, BRAF V600E mutation and markedly reduced HLA-I on tumor cells). Tumor samples obtained at surgical resection also revealed PD-L1 expression on tumor cells and a substantial fraction of tumor-infiltrating PD-1 ⁺ NK cells. These findings raise the hypothesis that PD-1/PD-L1 blockade may have contributed to NK-cell-mediated antitumor activity in the context of markedly reduced HLA-I expression. Furthermore, assessing PD-L1 and HLA-I expression on tumor cells at diagnosis could help identify patients who could benefit most from pembrolizumab treatment, alone or combined with inhibitors of HLA-I-specific inhibitory NK receptors. These observations provide a rationale for further investigation in larger and multicenter patient cohorts.

Programmed cell death protein 1 (PD-1, CD279) is a pivotal inhibitory immune checkpoint receptor that plays a central role in maintaining immune homeostasis and peripheral tolerance. Originally characterized as a negative regulator of T-cell activation, PD-1 limits excessive immune responses and prevents autoimmunity, while its sustained expression under conditions of chronic antigen stimulation contributes to T-cell dysfunction and exhaustion. The discovery that blockade of the PD-1 pathway can restore anti-tumor immunity has revolutionized cancer therapy and established immune checkpoint inhibition as a cornerstone of modern oncology. Although PD-1 has traditionally been viewed as a key regulator of adaptive immunity, accumulating evidence indicates that its biological functions extend beyond T cells. In recent years, PD-1 expression has been identified in several innate immune cell populations, particularly Natural Killer (NK) cells, where it has emerged as an important modulator of effector functions, cytokine production, metabolic fitness, and antitumor activity. These findings have challenged the classical view of PD-1 biology and revealed unexpected similarities between NK-cell dysfunction and the exhausted phenotype described in chronically stimulated T cells. In the tumor microenvironment, PD-1 expression on NK cells has been associated with impaired cytotoxicity and reduced immune surveillance, suggesting that NK cells may also represent relevant targets of PD-1-mediated immunosuppression. At the same time, the mechanisms regulating PD-1 expression and signaling in NK cells appear to differ, at least in part, from those operating in T lymphocytes, highlighting the complexity of this pathway across distinct immune cell subsets. In this review, we summarize the current knowledge of PD-1 biology, from its established role in T-cell regulation to its emerging functions in NK cells. We discuss the molecular mechanisms governing PD-1 expression and signaling, its contribution to immune dysfunction in cancer and chronic diseases, and the potential implications of targeting the PD-1 axis to enhance both adaptive and innate antitumor immunity.

Objective:To provide an updated overview of how molecular pathology is reshaping diagnostic, prognostic, and therapeutic paradigms in gynecologic oncology and redefining the role of the gynecologic pathologist in the era of precision medicine.Mechanism:Advances in next-generation sequencing (NGS) and large-scale genomic initiatives have enabled comprehensive molecular characterization of endometrial, ovarian, and cervical carcinomas. The integration of genomic, immunophenotypic, and clinicopathologic data supports a multidimensional diagnostic framework that refines risk stratification and guides targeted and immune-based therapies. Emerging tools, including liquid biopsy, digital pathology, and artificial intelligence (AI), are further advancing the clinical integration of molecular data.Findings in Brief:In endometrial carcinoma (EC), molecular classification complements and often surpasses morphology-based systems by providing more accurate prognostic assessment and informing therapeutic decision-making. In ovarian carcinoma, assessment of breast cancer susceptibility gene (BRCA) alterations and homologous recombination deficiency (HRD) has become central to personalized treatment strategies. In cervical carcinoma, although persistent high-risk human papillomavirus (HPV) infection remains the principal oncogenic driver, additional genomic alterations are increasingly being incorporated into the management of advanced disease. Furthermore, NGS enables the identification of germline alterations associated with hereditary cancer syndromes, thereby reinforcing the pathologist’s role in identifying familial cancer predisposition and supporting appropriate referral for genetic counseling.Conclusions:Molecular pathology is driving the transition toward an integrated, biology-driven model of gynecologic oncology, positioning the gynecologic pathologist as a key clinical integrator in precision medicine.

Lactate is recognized as a crucial signalling molecule within the tumor microenvironment, where it shapes immune responses by modulating various cell populations, including T cells and macrophages. However, its effect on natural killer (NK) cells, key effectors of early antitumor immunity, remains poorly understood. This study investigates how intratumoral lactate accumulation affects NK cell function in breast cancer, a neoplasm characterized by elevated glycolytic flux. An in-silico analysis of 882 breast cancer patients revealed that high lactate metabolism is inversely correlated with NK cell activation genes and is associated with poor prognosis. To corroborate these findings, NK cells from healthy donors were cultured under lactate-rich or control conditions. Lactate exposure impaired NK cell proliferation, downregulated activation markers and cytotoxic molecules, disrupted mitochondrial bioenergetics, and induced lipid accumulation, as demonstrated by flow cytometry, metabolic profiling, and Raman spectroscopy. Functional assays using microfluidic devices and degranulation tests revealed that lactate-exposed NK cells exhibited reduced chemotaxis and diminished cytotoxicity against MCF-7 and MDA-MB-231 breast cancer spheroids, accompanied by decreased CXCL9 and CXCL10 production. Pharmacologic inhibition of lactate transport, via Syrosingopine or MSC-4381 and AZD3965 combination, restored NK cell cytotoxicity in tumor co-cultures, as shown by increased NK cell degranulation, caspase-3/7–mediated tumor apoptosis, and spheroid shrinkage. Finally, GPR81 deletion mirrored these effects, enhancing NK cell activity. These findings identify lactate as a driver of NK cell suppression and highlight lactate transport and receptor targeting as a strategy to enhance NK cell–based immunotherapies in breast cancer and other lactate-rich tumors.

Photobiomodulation (PBM) has shown promising potential to enhance bone regeneration; however, its optimal delivery parameters and interactions with osteoconductive scaffolds remain insufficiently defined. This preclinical study is the first to incorporate a pilot dosimetry evaluation to standardize 980-nm PBM delivery and ensure that effective irradiance reached the target surface of critical-size calvarial defects in mice. The primary aim was to evaluate the effectiveness of this novel 980-nm PBM protocol delivered using either flat-top (FT) or standard Gaussian (ST) handpieces in enhancing bone regeneration in critical-size defects (CSDs), both with and without Bio-Oss^® grafting. A total of 120 adult mice were allocated into twelve experimental groups (n = 10 per group): untreated (control), Bio-Oss^® alone, PBM alone, and PBM combined with Bio-Oss^®, using either FT or ST handpieces, and evaluated at 30 and 60 days. Animals received 980 nm irradiation at 0.6 W (nominal power output–set on laser interface) in continuous-wave mode for 60 s, three times per week, for two consecutive weeks. Pilot dosimetry included power meter measurements to determine the therapeutic power reaching the defect surface area and temperature monitoring to ensure safe energy delivery. The dosimetry study demonstrated that, after accounting for the optical properties of mouse shaved skin and the Bio-Oss^® graft covered with Bio-Gide^® membrane, the effective irradiance reaching the base of the defect surface area was 1.131 W/cm² for the FT handpiece and 0.413 W/cm² for the ST handpiece. This dose was sufficient to induce significant regenerative effects. Histological, Masson’s trichrome, and immunohistochemical analyses for Runx2, OCN, GLI1, CD34, and CTSK were performed to characterize early and late osteogenic events. The combination of PBM and Bio-Oss^® significantly accelerated bone regeneration compared with PBM alone, with the FT handpiece producing the most uniform and advanced osteogenesis. PBM enhanced progenitor activation, osteoblast differentiation, angiogenesis, matrix deposition, and late-stage remodeling, demonstrating a synergistic effect with the scaffold, whereas Bio-Oss^® alone or defect alone showed limited early regenerative potential. These findings highlight the effectiveness of this novel standardized PBM dosimetry and uniform beam profile (FT), supporting their use as a foundation for future randomized controlled trials in craniofacial bone repair.

Early identification of the risk of malignant transformation in oral potentially malignant disorders (OPMDs) is critical for improving outcomes in oral squamous cell carcinoma (OSCC). This comprehensive review examines immunological biomarkers obtained from minimally invasive oral cytobrush (OCB) specimens for the early detection of OSCC within a precision medicine framework. The objectives were to (1) identify and characterise key immunological biomarkers associated with early oral carcinogenesis; (2) evaluate the diagnostic utility of OCB sampling for detecting these biomarkers; and (3) explore the potential of OCB-based profiling to support personalised screening and patient management. The review highlights the potential advantages of OCB compared with conventional diagnostic methods, as reported in the literature, particularly its ability to capture early malignant changes through immunological analysis. Evidence is discussed for biomarker pathways related to cell-cycle and differentiation dysregulation (p53, Ki-67, CKs), inflammation-driven epithelial transformation (IL-1β, IL-6, IL-8, TNF-α), and immune suppression and checkpoint activation (PD-L1, B7-H6). OCB provides reliable and patient-friendly cyto-salivary samples that are suitable for immunological and molecular analyses. Aberrant biomarker expression detected in OCB specimens correlates with epithelial dysplasia and reflects early non-invasive neoplastic transformation, supporting the diagnostic value of integrated biomarker panels. Overall, OCB-based immunoanalysis represents a practical, non-invasive approach for the early detection of OSCC. Emerging technologies, including AI and multi-omics approaches, may further support the precision and predictive values of immunological analysis for OSCC. When combined with relevant biomarker pathways reflecting tumour biology and host immune responses, this strategy could offer a strong foundation for precision-medicine screening. It may also support personalised monitoring in patients with OPMDs.

Innate lymphoid cells type 2 (ILC2s) are key regulators of tissue homeostasis and inflammation. In cancer, ILC2s can exhibit pro-tumoral functions by increasing the myeloid derived suppressor cells (MDSC)/T-cell ratio. Nevertheless, the upstream ILC2 triggers remain poorly defined. Here, we identify nerve growth factor (NGF) as the driver of ILC2 pro-tumoral functions in patients with bladder cancer. We show that ILC2s express the NGF receptor TrkA and respond to NGF by secreting type-2 cytokines. In the tumor microenvironment, NGF-producing mast cells accumulate and activate ILC2s to induce regulatory T cells (Tregs), ultimately fostering tumor growth. In patients, NGF levels inversely correlate with survival in ILC2-rich tumors, underscoring the clinical significance of this axis. In vivo administration of a selective TrkA inhibitor improves survival in orthotopic tumor-bearing female mice and sensitizes them to immune checkpoint blockade (ICB). Overall, we identify NGF as an ILC2 activator that shapes pro-tumoral ILC2 functions. The blockade of TrkA ⁺ ILC2s might represent a targetable strategy to improve survival, particularly in ICB-resistant patients.

Background: Ovarian cancer (OC) is the fifth leading cause of cancer-related death among women, with High-Grade Serous Ovarian Carcinoma (HGSC) representing the most aggressive and prevalent subtype. Despite promising results in other malignancies, immune checkpoint blockade has shown limited efficacy in HGSC, highlighting the need for alternative immunotherapeutic targets. Methods: We conducted an integrated analysis combining multiparametric flow cytometry, RNA sequencing, multiplex immunohistochemistry, and functional assays to characterize NK cells isolated from peripheral blood, peritoneal fluid, primary tumor tissue, and metastases in 60 HGSC patients. Results: We identified a distinct population of PD-1⁺ NK cells enriched in HGSC tumors and metastatic sites but absent in healthy donors. These cells, characterized by a CD56 ^dim NKG2A⁺KIR⁺/⁻NKp46⁺CD57 ^low phenotype, displayed impaired cytotoxicity against autologous HGSC targets, correlating with poorer prognosis. Crucially, this dysfunction was reversible upon combined blockade of PD-1/PD-L1, NKG2A, and KIRs. Spatial and molecular profiling revealed that these cells localize within PD-L1⁺/HLA-E⁺ tumor niches, suggesting that immune suppression is spatially and molecularly coordinated. Transcriptomic analysis confirmed their altered functional state and highlighted actionable checkpoint targets. Conclusions: Our findings uncover a previously underappreciated population of dysfunctional PD-1⁺ NK cells in HGSC and demonstrate that their suppression is reversible through combinatorial checkpoint inhibition. These insights support the development of spatially-informed, NK-targeted immunotherapies for HGSC patients, particularly those resistant to T cell-based strategies.

Background: Natural killer (NK) cells are innate lymphocytes endowed with potent cytotoxic activity. The presence of tumor-associated NK cells has been correlated with better prognosis in several solid tumors including colorectal cancer (CRC). This malignant disease is the second cause of cancer death worldwide and is in urgent need for novel approaches to improve current immunotherapies. Since CRC microenvironment can induce NK cell dysfunction and hinder cancer control, understanding tumor-associated NK cell features is mandatory to fully unlock their immunotherapeutic potential.Purpose: Our study aims at elucidating the molecular and functional characteristics of tumor-associated NK cells in CRC focusing on the expression of immune checkpoints that critically regulate NK cell function. We performed an in-depth cytofluorimetric analysis of tumor-associated NK cells obtained by tissue dissociation of samples derived from 80 CRC patients comparing tumor with matched tumor-free tissue and peripheral blood, stratifying patients by tumor stage or MSI/MSS condition. Tumor tissue was also analyzed by immunohistochemistry.Results: NK cells expressing immune checkpoints (i.e., KIR, NKG2A and TIM-3) were significantly enriched in tumor compared to tumor-free tissue, and an increase in PD-1⁺ NK cells was observed in tumors compared to peripheral blood and tumor-free tissue, indicating TME-induced modulation. Notably, tumor-associated PD-1⁺ NK cells characterized MSI rather than MSS CRC. In addition, tumor-associated NK cells also expressed tissue residency markers (CD103 and/or CD49a) and displayed a distinct profile also including the PD-1⁺ NK cell subset in MSI CRC, possibly representing NK cells recruited from circulation, retained in tumors, and reconfigured by TME signals. Importantly, tissue resident NK cells adequately expressed activating NK receptors and cytotoxic molecules.Conclusions: These results suggest, together with an increased PD-L1 expression on MSI tumor cells, that the efficacy of immunotherapies in MSI CRC based on PD-1/PD-L1 blockade could also rely on a superior anti-tumor potential of PD-1⁺ NK cells. Conversely, MSS CRC, in which tumor-associated PD-1⁺ NK cells are scarce, could benefit more from immunotherapies blocking NKG2A and/or KIRs. Thus, novel approaches based on NK cell features related to CRC type, fully exploiting circulating and resident NK cell anti-tumor activity, could be key to next-generation therapies.