Background: Due to the expanding role of immune checkpoint inhibition in the treatment of head and neck squamous cell carcinoma, understanding immunological processes in the tumor microevironment (TME) has strong translational importance. Though analytical methods for a comprehensive analysis of the immunological TME have constantly improved and expanded over the past years the prognostic relevance of immune cell composition in head and neck cancer TME largely remains ambiguous with most studies focusing on one or a small subset of immune cells.Methods: The overall survival (OS) of the TCGA-HNSC patient cohort comprising 513 head and neck cancer patients was correlated with a total of 29 different immune metrics including a wide spectrum of immune cell subpopulations as well as immune checkpoint receptors and cytokines using RNAseq based immune deconvolution analyses. The most significant predictors of survival among these 29 immune metrics were validated on a separate HNSCC patient cohort (n=101) using immunohistochemistry: CD3, CD20+CXCR5, CD4+CXCR5, Foxp3 and CD68.Results: Overall immune infiltration irrespective of immune cell composition showed no significant correlation with the patients’ overall survival in the TCGA-HNSC cohort. However, when focusing on different immune cell subpopulations, naïve B cells (p=0.0006), follicular T-helper cells (p<0.0001), macrophages (p=0.0042), regulatory T cells (p=0.0306), lymphocytes (p=0.0001), and cytotoxic T cells (p=0.0242) were identified as highly significant predictors of improved patient survival. Using immunohistochemical detection of these immune cells in a second independent validation cohort of 101 HNSCC patients, we confirmed the prognostic relevance of follicular T helper cells, cytotoxic T cells and lymphocytes. In multivariable analysis, HPV negativity and advanced UICC stages were identified as additional prognostic biomarkers associated with poor outcome.Conclusion: Our study highlights the prognostic relevance of the immunological tumor environment in head and neck cancer and demonstrates that a more detailed analysis of immune cell composition and immune cell subtypes is necessary to accurately prognosticate. We observed the highest prognostic relevance for lymphocytes, cytotoxic T cells, and follicular T helper cells, suggesting further investigations focusing on these specific immune cell subpopulations not only as predictors of patient prognosis but also as promising targets of new immunotherapeutic strategies.

Head and neck squamous cell carcinomas (HNSCC) have an overall poor prognosis, especially in locally advanced and metastatic stages. In most cases, multimodal therapeutic approaches are required and show only limited cure rates with a high risk of tumor recurrence. Anti-PD-1 antibody treatment was recently approved for recurrent and metastatic cases but to date, response rates remain lower than 25%. Therefore, the investigation of the immunological tumor microenvironment and the identification of novel immunotherapeutic targets in HNSCC is of paramount importance. In our study, we used tissue samples of n = 116 HNSCC patients for the immunohistochemical detection of the intratumoral and peritumoral expression of T cell exhaustion markers (PD-1, LAG-3, TIM-3) on tumor infiltration leukocytes (TIL), as well as the expression level of stromal senescence markers (IL-8, MMP-3) on tumor-associated fibroblasts. The clinical parameter of the vitamin D serum status as well as the histopathological HPV infection status of the tumor was correlated with the expression rates of the biomarkers and the overall patient survival. An increased peritumoral and intratumoral expression of the biomarkers PD-1 and TIM-3 significantly correlated with improved overall patient survival. A high peritumoral expression of LAG-3 correlated with better overall survival. A positive HPV tumor status correlated with a significantly elevated expression of PD-1 and TIM-3. Biomarkers of stromal senescence showed no influence on the patient outcome. However, the vitamin D serum status showed no influence on patient outcomes or biomarker expressions. Our study identified PD-1, LAG-3, and TIM-3 as promising targets of a therapeutic strategy targeting the tumor microenvironment in HNSCC, particularly among HPV-positive patients, where a higher expression of these checkpoints correlated with an improved overall survival. These findings support the potential of antibodies targeting these immune checkpoints to enhance treatment efficacy, especially in the context of bispecific targeting.

Background: DEK::AFF2 fusion-associated carcinomas of the sinonasal tract are exceedingly rare, with fewer than 100 cases reported worldwide, but probably underrecognized. Recently classified by the WHO as a distinct provisional subtype of non-keratinizing squamous cell carcinoma, these tumors pose significant diagnostic and therapeutic challenges. Their histological resemblance to inverted papillomas and their bland histology in most cases often leads to misdiagnosis, while their aggressive behavior underscores the need for a tailored treatment approach.Case presentation: We report two cases of DEK::AFF2 fusion-associated carcinomas managed at Saarland University Medical Center. The first case involved a 46-year-old woman who initially presented with recurrent sinonasal inverted papilloma, confirmed through multiple surgical interventions over nearly a decade. In 2023, reevaluation and genetic analysis revealed a DEK::AFF2 fusion. The patient demonstrated an exceptional response to three cycles of neoadjuvant gemcitabine and cisplatin, achieving complete remission on MRI restaging. This allowed a shift to definitive chemoradiotherapy, with sustained disease-free status confirmed by a PET-CT three months post-treatment in July 2024. The second case involved a 66-year-old woman presenting with recurrent inverted papilloma affecting the sinonasal and tympanic regions. Despite multiple surgeries, malignant transformation to invasive squamous cell carcinoma occurred, with lymph node metastasis and intracranial spread. A combined otolaryngological and neurosurgical approach was undertaken, but the disease progressed. The patient passed away in January 2020, with postmortem review of the prior histology and genetic analysis confirming DEK::AFF2 fusion carcinoma that showed bland-looking papilloma-like morphology in the initial specimens and later a high-grade cytology indicating biological progression to poorly differentiated carcinoma.Conclusion: These cases highlight the aggressive nature of DEK::AFF2 fusion-associated carcinomas and the critical role of genetic profiling in diagnosis and management. The exceptional, first ever reported response to neoadjuvant chemotherapy in one case underscores the potential for personalized treatment strategies, warranting further investigation into targeted therapies for this rare malignancy.

Background: Head and neck squamous cell carcinomas (HNSCC) pose significant clinical challenges, particularly in high‐risk cases with positive lymph node status. Current prognostic biomarkers are often costly and methodologically demanding. In this regard, histomorphological biomarkers such as tumor buds (TB) and poorly differentiated clusters (PDC) represent promising, cost‐effective prognostic indicators that are relatively straightforward to implement.Methods: The prognostic significance of TB and PDC, in conjunction with stromal tumor‐infiltrating lymphocytes (sTILs) and the tumor‐stroma ratio (TSR), was evaluated in a cohort of 50 high‐risk, nodal‐positive HNSCC patients. Histomorphological features were assessed using standard hematoxylin and eosin (H&E) staining, while HPV association and PD‐L1 expression were determined by means of immunohistochemistry (IHC) and/or PCR. All variables collected were subsequently correlated with traditional histopathological and clinical parameters. Finally, a novel scoring system incorporating TB and PDC was developed, and its association with overall survival (OS) was analyzed.Results: TB and PDC both demonstrated a significant impact on patients' OS (TB Log‐rank test, p = 0.0499, PDC Log‐rank test, p = 0.0235). A novel scoring system based on these features had strong association with patients' OS (Log‐rank test, p = 0.0200) in contrast to the conventional and routinely performed grading system, which evaluates the degree of differentiation within neoplastic cells (Log‐rank test, p = 0.3325). PD‐L1 expression was not associated with TB and PDC formation. HPV‐negative status was associated with a higher number of tumor buds.Conclusion: This study reveals the potential prognostic value of TB and PDC in high‐risk HNSCC, which may offer a practical and cost‐effective alternative to traditional markers. Our proposed practicable and straightforward employable scoring system significantly correlates with OS, suggesting its potential benefit in clinical practice. These findings advocate for further validation to enhance prognostic accuracy and guide treatment strategies in HNSCC.

Salivary gland carcinomas are rare, heterogeneous, and often resistant to conventional treatments, highlighting the need for new therapeutic strategies. This retrospective study evaluated the expression of three immunologically relevant biomarkers—PRAME, FOLR1, and CLDN18.2—as potential therapeutic targets in salivary gland carcinomas. Tumor samples from 54 patients with seven histological subtypes treated at Saarland University Medical Center between 2013 and 2023 were analyzed using immunohistochemistry and scored with the Immunoreactive Score. PRAME was strongly expressed in 89% of cases, while FOLR1 and CLDN18.2 showed markedly lower expression at 41% and 6%, respectively. High PRAME expression was significantly associated with the presence of distant metastases, regardless of histological subtype. Patients with low PRAME expression tended to have improved overall survival, as indicated by Kaplan–Meier analysis and Log‐Rank testing. These findings suggest PRAME as a promising immunotherapeutic and diagnostic target for salivary gland carcinomas, particularly through T‐cell‐based therapies already under investigation in other malignancies such as acute myeloid leukemia. Further preclinical studies are needed to validate the functional and therapeutic significance of PRAME, FOLR1, and CLDN18.2 in this context.

Background: The role of immune checkpoint inhibition in treating head and neck squamous cell carcinoma (HNSCC) is expanding, yet response rates to PD-L1 therapy remain inconsistent and generally poor. Although several studies have examined heterogeneous intratumoral PD-L1 expression, the disparity in response to PD-L1 therapy between primary tumors and their associated lymph node metastases remains unclear. Methods: Primary tumor samples and two matching lymph node metastases were obtained from a cohort of 50 patients and immunohistochemically stained with a PD-L1 antibody. PD-L1 expression, assessed using the combined positive score (CPS) and tumor proportion score (TPS), and immune infiltration, measured with an immunoreactive score (IRS), were compared between the primary tumor and lymph node metastases. These measures were then correlated with other histopathological and clinical features. Results: PD-L1 expression, evaluated by CPS and TPS, showed no significant differences between the primary tumor and matched lymph node metastases. Discordance relative to established regulatory cut-offs was observed in a subset of patients, affecting 18% (CPS; 95% CI, 8.0–30.0%) and 4% (TPS; 95% CI, 0.0–10.0%) of cases. CPS and TPS values were not influenced by primary tumor subsite or HPV status. Conversely, immune infiltration measured by IRS was significantly affected by primary tumor subsite location. Both HPV tumor status and primary tumor subsite were statistically significantly associated with overall survival. Conclusions: Our findings highlight variability in PD-L1 expression in HNSCC and may offer context for differential responses of primary tumors and lymph node metastases to immune checkpoint therapy reported in recent clinical studies. These observations support the need for a more comprehensive characterization of PD-L1 expression across tumor sites in head and neck cancer. Further investigation is required to determine whether, and in which settings, reassessment of PD-L1 status in metastatic lesions—including lymph node metastases—may provide additional clinically relevant information when initial testing does not meet established therapeutic cut-offs.