Lymphoid aggregates represent the earliest and most immature form of TLSs. These structures are characterized by CD20+ B-cell clusters that lack follicular dendritic cell networks (CD21-) and absence of CD23 expression (CD23-). The immunophenotypic profile is CD20+CD21-CD23-, indicating neither architectural organization nor GC functionality. They consist of loosely organized B and T lymphocytes without defined compartmentalization or specialized stromal support structures.

Non-GC TLSs demonstrate intermediate maturation with organized architecture but lack functional GC activity. The defining immunophenotypic signature is CD20+CD21+CD23-. The presence of CD21+ follicular dendritic cells here indicates structural organization with formation of follicular dendritic cell networks, but the absence of CD23 confirms lack of GC functionality. These structures exhibit spatial segregation of B-cell and T-cell zones but do not support active GC reactions including somatic hypermutation and affinity maturation.

GC-like TLS represent the most mature, functionally active form with complete GC organization and functionality. The immunophenotypic profile is CD20+ CD21+ CD23 +. These structures contain CD23+ follicular dendritic cells within well-developed GCs, indicating full functional capacity for B-cell selection and antibody affinity maturation. Additional GC markers include BCL6+ proliferating B cells (a transcription factor essential for GC formation), Ki67+ cells (marking active proliferation with distinct dark and light zones), and BCL2- GC B cells (distinguishing them from naïve B cells).

TLS assessment employs multiple complementary methodologies, each with distinct advantages and limitations. H&E-based identification remains the foundation of TLS detection, relying on recognition of dense lymphoid aggregates with or without visible GCs (8, 9). This approach enables initial screening but requires validation with immunohistochemistry (IHC) for accurate maturation staging. Standard IHC panels typically include CD20 (B cells), CD3 (T cells), CD21 (follicular dendritic cells), CD23 (GC marker), BCL6 (GC B cells), and Ki-67 (proliferation marker) (9, 10). A streamlined approach using H&E staining combined with CD23 IHC has demonstrated high interrater agreement (kappa 0.90) for maturation assessment and can be applied across all specimen types (9).

Quantification approaches vary substantially across studies, contributing to heterogeneity in prognostic comparisons. Common metrics include TLS density (TLS per mm² of tumor area), TLS area fraction (percentage of total tissue occupied by TLS), and binary presence/absence scoring (8, 11, 12). Gene expression signatures offer an alternative or complementary approach, with validated panels including 12-chemokine signatures (CXCL13, CCL19, CCL21), 7-gene signatures (CXCL13, CCL19, MS4A1, LTB, CD37, CORO1A, IKZF1), and broader 17–23 gene panels incorporating B-cell, T-cell, and dendritic cell markers (13–15). These transcriptomic approaches enable TLS assessment in archival samples lacking tissue for IHC but may not capture spatial organization or maturation status as precisely as histological methods. The lack of standardization across these methodologies significantly impacts cross-study comparisons and limits clinical implementation, highlighting the need for consensus guidelines on TLS assessment in genitourinary malignancies (9, 14).

Some studies employ a morphology-based classification distinguishing early TLS (E-TLS), primary follicle-like TLS (PFL-TLS), and secondary follicle-like TLS (SFL-TLS) (7, 16–18). Secondary follicle-like TLSs resemble mature GC-like TLS with visible GCs on H&E staining or CD23+ follicular dendritic cells. This classification focuses on the presence of GCs as the critical distinguishing feature of mature TLS.

The maturation stage directly correlates with functional capacity and clinical outcomes. Mature GC-like TLS with functional GCs demonstrate superior prognostic value compared to immature lymphoid aggregates, particularly in predicting immunotherapy response (19). This reflects the enhanced capacity of mature TLS to support local adaptive immune responses, including B-cell affinity maturation, plasma cell differentiation, and generation of tumor-reactive antibodies (20, 21).

In PCa, the presence of TLSs has gained interest due to their role in immunomodulation, therapy, and prognosis (24, 25). In localized PCa, TLSs can be found in peri-tumoral or intra-tumoral regions (24). These structures are enriched in immune-active gene expression, containing T-cell and B-cell related clusters and MHC class I/II (24). The presence of TLSs is also negatively associated with immunosuppressive profiles, such as T-regulatory cells and myeloid-derived suppressor cells; suggesting a role in counteracting immune evasion (24).

TLS formation in PCa may be driven by chemokines and cytokines such as chemokine (C-X-C motif) ligand (CXCL)-13 and interleukin (IL)-7, which promote lymphoid cells recruitment and the formation of HEVs (1). Additionally, TLS maturation and immunologic support are regulated through lymphotoxin-b receptor signaling, which coordinates immune-stromal communication and chemokine production (26). Classically, PCa’s are classified as immunologically “cold”, but TLS-positive tumors can have CD8+ T-cell infiltration, which is related to improved outcomes and reduced recurrence (27). This antitumor potential is enhanced in mature TLSs that contain follicular dendritic cells and GCs, by supporting antigen presentation and B-cell activation (6).

The presence of TLSs is linked to greater CD8+ T-cell infiltration, higher Th1 responses, and reduced Treg density, which work to slow tumor growth and increased immune-mediated cytotoxicity (5, 27–29). However, in some cases, poorly organized or immature TLSs may fail to create an appropriate immune response and instead be a niche for suppressive immune cell clusters (24, 30). Thus, TLSs maturity appears to play a role in determining their effects on disease progression. The physical location of TLSs within PCa’s also plays a role in modulating the TME. Peri-tumoral TLSs are related to antigen presentation pathways, while intra-tumoral TLSs are more associated with T-cell-related activity (24). Generally, TLSs have been associated with better prognosis across many malignancy types, and preliminary data suggest similar implications in PCa, opening the door for the development of new prognostic and therapeutic modalities (6).

TLSs are less frequently found in metastatic PCa, especially in bone and liver metastases (30). The sites of metastasis are dominated by immunosuppressive cytokines and are characterized by the absence of TLSs and reduced antigen expression, contributing to immune escape and resistance to treatment (30, 31).

The absence or dysfunction of TLSs in metastatic disease contributes to the poor response rates observed with immune checkpoint inhibitors in PCa. For example, trials using PD-1 and CTLA-4 blockade have shown modest results, possibly due to a lack of organized immune zones within the tumor or low T-cell infiltration (31). Therefore, induction of TLSs formation through cytokine modulation or LTbR activation may improve immune recognition and decrease resistance to immunotherapy (1, 6, 26, 27).

Another promising use of TLSs lies in the heterogeneity of these structures in PCa, which highlights the need for TLS-based stratification. Patients with high TLSs density and mature structures may benefit from immunotherapy, whereas those lacking them might require TLSs-priming strategies before checkpoint inhibition. As a result, TLS profiling could be used as a guide to optimize treatment strategies (6, 24). Of note, while most of the existing data highlight their protective role, certain subsets of these TLSs may facilitate tumor survival (27, 30). Immature TLSs or those with immunosuppressive cellular environments via infiltration of T-regs and M2 macrophages, which reduce the efficacy of checkpoint blockade and other immune-based therapies, may promote disease progression (27, 30, 31). Therefore, it is essential to understand the cellular composition and maturation stage of TLSs. To achieve this, research has proposed developing a staging system for TLSs from early aggregates to structures with fully functional GC, which may help predict outcomes more accurately (1, 6).

Looking forward, a multi-modal approach combining TLS induction, immune checkpoint inhibition, and androgen deprivation therapy may offer synergistic benefits in the treatment of PCa’s. This would address both the immunosuppressive TMEs and the hormonal dependence of PCa’s, while promoting immune infiltration and memory T-cell generation (26, 31).

While TLSs are present in early-stage bladder cancer, they tend to be less mature and organized in NMIBC, potentially due to less chronic inflammation in early stages of the disease (1, 36). Spatial transcriptomic analysis has provided further insight, identifying the colocalization of CXCL13⁺ CD4⁺ T-cells and CXCR⁺ NR4A2⁺ B-cells within TLSs regions, highlighting the CXCL13-CXCR5 axis as a key mechanism in TLS formation (15). Mature TLS, marked by structured, well-defined, B-cell zones and follicular dendritic cell networks, were more commonly observed in MIBC tumors, underscoring the progressive maturation in TLSs in the setting of advancing disease (15).

The presence and density of TLSs in NMIBC cancer has demonstrated a strong association with improved response to Bacillus Calmette-Guerin (BCG) therapy. In a cohort analysis, 62.5% of patients with high TLS density achieved a complete response to BCG, compared to only 27.3% in the low TLS density group, further cementing the value TLSs have in achieving favorable therapeutic responses (32). Additionally, NMIBC patients whose tumors had a high TLSs signature scores experienced significantly longer recurrence-free survival (RFS) and had greater infiltration of CD8⁺ T cells and B cells, both critical for anti-tumor immunity (36). These findings may be attributed to TLSs enhancing BCG efficacy by enabling a coordinated immune response through the recruitment and activation of B-cells, dendritic cells, and T cells within the TME (1).

TLSs have also emerged as promising prognostic biomarkers in NMIBC. Although TLSs were less frequently observed in NMIBC compared to muscle-invasive disease, their presence still correlated with improved survival outcomes, including in BCG-treated patients, though this did not reach statistical significance in some analyses (15). Regardless, TLS signature scores were independently associated with increased RFS and progression-free survival (PFS) in NMIBC patients and high TLS scores were linked to better disease-free survival (DFS) across three independent bladder cancer cohorts (37, 38). Lastly, retrospective analyses also identified TLSs as a biomarker of reduced recurrence and progression risk in NMIBC (1).

In MIBC, TLSs are more prevalent and structurally mature compared to those in early-stage disease; supporting the idea of increased density and organization with advanced tumor biology (39). At the immunological level, TLSs were positively correlated with CD8⁺ T cells, dendritic cells, and M1 macrophages, and negatively associated with immunosuppressive M2 macrophages and T-regs, supporting the evidence that mature, functioning TLSs contribute to a tumor-suppressive microenvironment (40). In a study by Ligon et. al., TLSs were observed in 60% of MIBC tumors, predominantly as mature follicular TLS (FL-TLS) characterized by CD21⁺ follicular dendritic cells and GC-like architecture (41). Additionally, a high TLS signature score in patients receiving anti-PD-1/PD-L1 therapy resulted in improved objective response rate (ORR) and (PFS), reinforcing the evidence of the significant role these TLSs play therapy response (36).

TLSs in MIBC are strongly associated with an immune-inflamed TME characterized by enhanced immune cell infiltration and functional activation. Correspondingly, gene expressions within TLS-enriched zones showed elevated levels of effector molecules such as GZMA, IFNG, and PDCD1, and signatures of tumor-reactive T-cells were significantly increased in TLS-positive MIBC samples (15). Plasma cell numbers were also increased in TLS-positive environments, and secreted immunoglobulin heavy constant gamma (IGHG)-1 and IGHG3 which are immunoglobulin isotypes associated with antibody-dependent cellular phagocytosis (15).

In MIBC, TLS-rich tumors also exhibit increased densities of CD8⁺ T cells, CD20⁺ B cells, and DC-LAMP⁺ dendritic cells, suggesting a robust immune landscape and a highly inflamed immune tumor milieu (15, 37). Immune cell densities were greatest within TLSs regions of interest and decreased with distance from TLSs, emphasizing their localized immunological activity (42). Furthermore, follicular TLSs (FL-TLSs) contained higher B-cell densities and occupied larger spatial domains than early TLSs (E-TLSs), further supporting the relation between structural maturity and adequate function (42). These structurally mature TLSs seen in MIBC, are also associated with elevated CD8⁺ T-cell infiltration, which further highlights the immune-inflamed tumor phenotype (1). Together, these organized structures promote antigen presentation via follicular dendritic cells and mature dendritic cells and coordinate T and B-cell zones, thereby establishing a localized immune-responsive niche within the TME (39).

The presence of TLSs and their associated genetic signatures have been linked to favorable responses to immune checkpoint blockade in MIBC. In the IMvigor210 cohort, patients with higher TLSs gene signature scores exhibited significantly improved overall survival (OS) following anti-PD-L1 therapy (15). A 7-gene TLS-specific signature, comprising CXCL13, CCL19, MS4A1, LTB, CD37, CORO1A, and IKZF1, was developed to stratify patients by their response to checkpoint inhibitors (15). Similarly, TLS score was a strong predictor of complete or partial response among anti-PD-L1-treated patients in IMvigor210 (38). Notably, patients categorized as TLS-high-PMN-MDSC-low demonstrated the best prognoses, while TLS-low-PMN-MDSC-high had the poorest outcomes (42). TLS signatures, including 12-chemokine and CXCL13-based models, were also associated with enhanced survival in the IMvigor210 trial (42). Multiple large-scale studies, such as the IMvigor210 cohorts, have confirmed elevated TLS gene expression in responders to PD-1/PD-L1 blockade (1). Clinical evidence continues to demonstrate that TLS-rich tumors in bladder cancer respond more favorably to checkpoint blockade therapy, and high TLS signature scores are also associated with elevated expression of immune checkpoint molecules and superior anti-PD-L1 therapeutic response (39, 40).

TLS structures also appear to positively influence therapeutic responsiveness within the context of neoadjuvant therapies. In the BLASST-1 trial, which evaluated neoadjuvant nivolumab, gemcitabine, and cisplatin followed by radical cystectomy in patients with MIBC, transcriptome-wide profiling of 37 pretreatment transurethral resection of bladder tumor (TURBT) specimens was performed to identify signatures predictive of pathological response rate (PaR) and pathological complete response (PCR) (43). Higher immune activation signatures (which serve as a proxy for TLS presence) and other immune-rich signatures correlate with better PaR in the TME (43). These findings further support the role of TLS in establishing an immune-inflamed TME that enhances therapeutic sensitivity and contributes to improved treatment outcomes.

TLSs are found in both primary and metastatic bladder cancer, though those in metastases are typically less mature and organized (44). To better characterize TLS in this context, a seven-gene TLS-related signature, comprising CXCL13, CCL19, CD79A, CD27, MS4A1, LTB, and SELL, was developed using LASSO regression to predict TLS presence and immune activity across primary and metastatic disease (38).

TLSs play a key role in systemic immune responses in bladder cancer. A conserved immune profile, including TLS-related gene signatures, is seen across matched primary and metastatic tumors, suggesting TLSs contribute to a unified systemic immune phenotype (44). In metastatic blader cancer, TLS score was also found to positively correlate with increased CD8⁺ T cell infiltration, dendritic cell abundance, and M1 macrophages, B-cell receptor signaling genes, class-switched immunoglobulins, and antigen presentation components, while showing a negative correlation with M2 macrophages and Tregs, indicating a tumor-suppressive immune landscape (38, 41).

TLS-associated gene signatures, particularly those including CXCL13 and CCL19, have also been validated in multiple cohorts treated with PD-1/PD-L1 blockade, further underscoring their role in mediating antitumor immunity at both local and systemic levels (33). Patients with high TLS gene expression signatures in metastatic lesions also exhibited similar r benefits resulting in longer PFS following checkpoint blockade therapy (44). Integration of TLSs mapping with digital pathology techniques enables spatial identification of immune-enriched regions that correlate with treatment response and survival outcomes (36). Among patients treated with anti-PD-1 or PD-L1 agents, those with high TLS scores demonstrated higher ORR (42% vs. 19%) and longer OS (median not reached vs. 9.4 months) (37).

Given these findings, an immune scoring approach based on TLS high PMN-MDSC low has been proposed to help identify patients most likely to benefit from immune checkpoint blockade (42). Additionally, in support of translational strategies, induction of TLSs through cytokines such as LIGHT and CCL21 or via dendritic cell vaccines has shown preclinical efficacy in enhancing antitumor immunity (33).

Bladder cancer has been a major focus of research investigating how immune checkpoint therapies may drive TLS development. One example is the phase 1b NABUCCO trial, which evaluated neoadjuvant ipilimumab and nivolumab in patients with MIBC. The combination targets two key immune checkpoints, CTLA-4 and PD-1. During the study, 24 patients were treated with two doses of ipilimumab followed by nivolumab. It was found that 46% of participants showed no viable tumor at the time of cystectomy post-treatment (45). Upon histological and molecular analysis, a subset of specimens demonstrated apparent TLS formation, which was defined by dense lymphocytic aggregates, the presence of follicular dendritic cells, and the evidence of B-cell activation and immune infiltration (45). The finding supported that the checkpoint blockade may not only enable the formation of TSLs within tumors but also elicit antitumor immunity (45). Within the context of dual checkpoint blockade, another study analyzed the effects of durvalumab/tremelimumab combination in cisplatin-ineligible patients. The study showed promising results with a viable tumor rate of 37.5% and a downstaging rate of 58% (46). While TLSs were not directly quantified, upregulation of B-cell associated genes was observed in responding tumors using transcriptomic analysis, consistent with TLS activity (46).

Currently, ongoing trials aim to better understand and enhance TLS responses. The SURE-01 trial evaluates pembrolizumab, a PD-1 inhibitor, as neoadjuvant therapy in cisplatin-ineligible patients, incorporating tissue analysis to assess changes in TLS density and maturity (47). The NURE-Combo trial studies nivolumab with nab-paclitaxel to determine whether this combination induces immune infiltration and promotes TLS formation (48, 49). The SURE-02 trial tests pembrolizumab with sacituzumab govitecan, targeting Trop-2, to evaluate the effects of combining immune checkpoint inhibition with targeted cytotoxic therapy on TLS induction and maintenance (50).

In a model of autoimmune orchitis, TLSs resembling secondary lymphoid structures consisting of B-cells, T-cells, follicular dendritic cells, and HEVs have been observed; indicating local antigen processing and adaptive immune activity (51). In these models, TLS formation in the testes was associated with exposure to spermatic antigens and characterized by upregulation of the lymphoid-organizing chemokines CXCL13 and CCL19 (51). Their formation was observed in areas adjacent to seminiferous tubule destruction, suggesting an immune response to localized antigen release (51). The reported upregulation of chemokines such as CXCL13 and CCL19 in these settings further provides evidence of lymphocyte aggregation and TLS maturation (51). Although these findings have been studied in the setting of an infectious process, they provide an opportunity to understand the role of TLSs in the testes.

TLSs have sporadically been identified in testicular tumors, especially seminomas, and they are typically associated with areas of dense lymphoid infiltration (1). Current data suggest that TLSs positive tumors play a role in local anti-tumor immune responses. Notably, the proximity of TLSs to intratumoral vessels and necrotic areas in seminomas suggests that antigen release from tissue damage may play a role in their formation (1). These findings suggest active local immune responses with organized B-cell and T-cell areas and mature dendritic cells (1). Unlike autoimmune orchitis models-where TLS formation is linked to spermatic antigen exposure and chemokine upregulation-TLS-like structures in seminomas appear near dense lymphoid infiltrates, though the exact factors driving their formation remain unknown (1, 51).

In testicular germ cell tumors (TGCTs), differences in TLS composition and presence are evident between seminoma and non-seminoma tumor subtypes. Of note, the presence of TLSs in seminomas without evidence of their suppression in the TME - along with their slow progression and favorable prognosis underscores the possible role of TLSs in slowing tumor progression and spread (1, 52). In contrast, non-seminomatous germ cell tumors - particularly embryonal carcinoma (EC)-exhibit a distinct immune profile, with evidence of active TLS suppression (52). Single-cell RNA sequencing has shown that high SERPINB9 expression in metastatic EC correlates with downregulation of TLS-related chemokines (CXCL13, IL-6, IL-6, and CCL5), suggesting a reduced capacity to recruit and organize immune cells into TLS-like structures (53). This immune suppression may hinder local anti-tumor immune response. SERPINB9 expression is also associated with increased tumor stemness features and greater metastatic potential, suggesting a broader role in shaping an immunosuppressive TME (53). Collectively, these findings highlight fundamental differences in TLS presence and immune organization between seminomas and non-seminoma TGCTs and highlights the need for further research in the role and significance of TLSs across testicular malignancies.

In testicular cancer, metastasis often occurs via lymphatic spread to retroperitoneal nodes - and the environment may either support or inhibit TLS formation depending on the tumor’s immune-modulating capabilities (53). SERPINB9 expression in metastatic sites has been associated with suppression of TLS-promoting chemokines, which may contribute to an immune-suppressive environment and resistance to immune checkpoint therapy (53). Their absence, especially in tumors expressing immunosuppressive mediators like SERPINB9, is associated with more aggressive tumor behavior and greater incidence of therapeutic resistance (53). This association serves as a promising area of study for predicting and monitoring tumor behavior and for developing new therapies.

TLS status could serve as a biomarker in testicular cancer management and immunotherapy planning. Moreover, modulating TLS-inducing chemokines and targeting suppressors genes may provide a foundation for TLS-focused immunotherapies. Further, insights from other malignancies suggest that strategies aimed at promoting TLS formation may hold promise in inducing immune responses in tumors with poor baseline immunogenicity (1, 52).

TLSs identified in early-stage renal cell carcinoma (RCC) correlates with enhanced infiltration of immune effector cells, particularly CD8⁺ cytotoxic T-cells and B-cells, promoting an inflammatory TME that may support both cellular and humoral antitumor immunity (54). Additionally, specific chemokines, most notably CXCL13, CCL19, and CCL21, have been identified as essential mediators in TLS formation, organization, and function. In ccRCC with high TLSs density, it was found that expression of these chemokines was significantly higher compared to TLSs negative tumors (54). The increase chemokine expression was also observed to correlate with higher immune effector cell infiltration (54). Interestingly, although TLSs are associated with a better prognosis, expression of CXCL13 was specifically associated with shorter progression-free and OS (14, 54).

In ccRCC, the presence of mature TLSs containing well-developed GCs is associated with significantly improved clinical outcomes (54). These patients with TLS-rich tumors experienced longer OS and progression-free survival (54). TLS-rich tumors were also associated with higher expression of immune checkpoint–relevant markers and greater predicted response scores to PD-1 and CTLA-4 inhibitors (54). These findings highlight the prognostic and therapeutic value of structurally mature and functional TLSs in ccRCC.

In their study, 57, analyzed matched primary tumors (PTs) and distant metastases (METs) from 47 patients with ccRCC. Digital spatial profiling was used to assess CD8⁺ Tcell distribution, co-expression of the exhaustion marker TOX, and to quantify TLS density (57). Most METs displayed an immune “cold” phenotype, defined by low densities of lymphocytes and TLSs (57). However, a small subset of METs classified as “inflamed” exhibited larger cumulative TLS areas and higher proportions of CD8⁺TOX⁺ T-cells compared to their matched PTs subset (57). At the same time, they observed that “hot” PTs, with dense CD8⁺ infiltration and increased CD8⁺TOX⁺ T-cells, were associated with shorter disease-free survival, suggesting that high immune cell presence may reflect T-cell exhaustion rather than effective immune control (57). These findings support the idea that immune features, including high TLSs density, may progress with advancing disease and evolve into dysfunctional units (57). In this context, TLSs may be used as highly informative biomarkers for stratifying disease progression, staging, and treatment options.

In localized penile squamous cell carcinoma (SCC), TLSs have been identified within the tumor parenchyma and surrounding stromal regions, indicating active local immune engagement (58). As seen in many other TLSs positive solid tumors, IHC analyses revealed that TLS-positive penile SCC exhibited higher infiltration of CD8+ T-lymphocytes and CD20+ B cells, along with more mature dendritic cells, compared to TLS-negative tumors (58). Of significant interest however, it was found that TLS positive penile SCCs were independently associated with better OS, regardless of tumor stage or nodal status, suggesting that localized immune responses within the TME significantly influences disease progression (58). Additionally, TLS-positive tumors exhibited upregulation of genes related to immune activation, including markers of cytotoxic T-cell activity such as granzyme B (GZMB) and interferon gamma (IFNG) (58). These tumors also had lower expression of immunosuppressive cytokines compared to their TLS-negative counterparts, suggesting a favorable environment for anti-tumor immune responses (58). These findings have been substantiated by a robust multicenter cohort of 165 penile SCC cases, in which patients with fewer TLSs demonstrated significantly worse OS with hazard ratio (HR) of 2.17 (95% CI: 0.94-5; P = 0.069). High B-cell immunoscore (reflecting CD20+ and CD138+ cell densities within and around TLS) was independently associated with improved OS with HR of 1.89 (95% CI: 1.18-3.03; P = 0.008) (59). Notably, high TLS diameter correlated with brisk lymphocytic infiltrate (OR = 2.24; 95% CI: 1.10-4.55; P = 0.021), and high B-cell immunoscores were strongly associated with mutated p53 profiles and enhanced immune activation (59, 60). Recent spatial transcriptomic analyses have further revealed that CD74+ B cells within TLS are enriched during early TLS formation and engage with naïve T cells through HLA-DRA-mediated interactions, activating key transcription factors (NFKB1, NFKB2, NFATC1, FOS, RUNX1) that enhance local immune responses and correlate with improved patient survival (61).

Further supporting this data, TLS-positive tumors were found to have lower serum levels of squamous cell carcinoma antigen (SCCAg), a biomarker that correlates to increased tumor burden and poor prognosis (62). These observations hint at the prominent role these structures have on penile SCC progression, and to their value as prognostic and therapeutic targets.

Lymphovascular invasion is a well-established adverse prognostic factor in penile SCC, and the presence of robust local immune responses may theoretically help limit distant spread (63). Supporting this statement, the study by Yi et al. (64) showed that weak local immune control at the primary tumor site in penile SCC is linked to early lymphatic spread and pelvic node metastasis (64). Although primary data are limited, in the context of the general findings by Yi et al. (64) and the established data supporting TLSs role in modulating antitumor immunity in solid tumors, it is worth noting the crucial role that TLSs may have in preventing penile SCC metastatic spread (64).

Beyond TLS-specific investigations, recent immuno-oncology studies have characterized the broader tumor immune microenvironment in penile SCC (60, 65, 66), revealing critical insights into immune checkpoint expression, tumor-immune interactions, and mechanisms of immune evasion that provide biological context for TLS-mediated immune control (67). Inflammation plays a crucial role in penile cancer development and progression, with the tumor immune microenvironment characterized by tumor-associated macrophages, cancer-associated fibroblasts, and tumor-infiltrating lymphocytes that produce pro-inflammatory cytokines and chemokines associated with tumor progression (65). The nuclear factor kappa B (NF-κB) pathway and secreted phosphoprotein 1 (SPP1) have been implicated in penile cancer pathogenesis, while elevated C-reactive protein (CRP) levels and neutrophil-to-lymphocyte ratio (NLR) have been identified as potential prognostic biomarkers, with high NLR (≥3.0) associated with advanced stage, lymphovascular invasion, and immunosuppressive tumor microenvironment characterized by increased N2 tumor-associated neutrophils and CD8+ T-cell exhaustion (65, 68).

In a large retrospective study of 152 penile SCC cases, brisk lymphocytic infiltrate was an independent predictor of improved overall survival and cancer-specific survival (HR for non-brisk/absent infiltrate: 2.22; P = 0.0023) (69). Multiplex immunofluorescence studies have revealed progressive T-cell exhaustion with advancing disease stage. An initial immune response in early locoregional disease (N1) with increased CD3+, CD4+, and CD8+ T-cell densities is followed by immune exhaustion in advanced disease (N2-3), marked by declining cytotoxic T-cell density, rising PD-L1 expression, and progressive replacement of anti-tumor M1 macrophages with pro-tumorigenic M2 macrophages (70). Additional studies demonstrate that PD-L1 expression at the invasive margin is significantly elevated in node-positive disease, TILs exhibit higher PD-1 expression compared to peripheral blood, and immune checkpoint molecules (PD-1, LAG3, TIM3) are co-expressed in high-grade tumors (66, 69, 71).

Furthermore, spatial analysis demonstrates that close clustering of M2 macrophages with tumor cells is associated with worse OS, recurrence-free survival, and cancer-specific survival, whereas bivariate clustering of CD3+CD4+ helper T cells with tumor cells correlates with improved outcomes, including in node-positive disease (70). These findings support the biological plausibility of TLS-mediated immune control, where TLS presence may counteract the immunosuppressive mechanisms (including T-cell exhaustion, M2 macrophage predominance, and checkpoint molecule upregulation) that characterize advanced penile cancer, thereby reinforcing the rationale for both TLS assessment as a prognostic biomarker and immune checkpoint inhibitor-based therapeutic strategies in this disease (72–75).

In metastatic penile SCC, TLSs have been identified within both inguinal and pelvic lymph nodes (58). IHC analyses demonstrated that metastatic lymph nodes containing TLSs exhibited higher densities of CD8+ T cells and CD20+ B cells relative to TLS-negative nodes, suggesting that TLS presence also sustain active local immune surveillance in metastatic lesions (58).

In penile squamous cell carcinoma (SCC), TLSs were observed more frequently in primary tumors from patients who did not have distant metastases, compared to those with metastatic disease (58). This pattern hints at an inverse relationship between TLS presence and the presence of metastatic lesions; though the concrete reason for this association remains unknown (58).

Overall, although current data are limited, these findings collectively suggest that TLS evaluation in penile SCC could provide valuable prognostic and therapeutic information for the management of these malignancies.