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Background: Survival rates of head and neck squamous cell carcinoma (HNSCC) have only marginally improved in the last decades. Hence there is a need for predictive biomarkers for long-time survival that can help to guide treatment decisions and might lead to the development of new therapies. The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway is the most frequently altered pathway in HNSCC, genes are often mutated, amplificated and overexpressed causing aberrant signaling affecting cell growth and differentiation. Numerous genetic alterations of upstream and downstream factors have currently been clarified. However, their predictive value has yet to be established. Therefore we assess the predictive value of p-mTOR, p-ERK and PTEN expression., Methods: Tissue microarrays (TMA's) of HPV-negative patients with oropharyngeal (n = 48), hypopharyngeal (n = 16) or laryngeal (n = 13) SCC, treated with primary chemoradiation (cisplatin/carboplatin/cetuximab and radiotherapy), were histologically stained for p-mTOR, PTEN and p-ERK. Expression was correlated to overall survival (OS), disease free survival (DFS) and locoregional control (LRC). Also p-mTOR was histologically stained in a separate cohort of HNSCC organoids (n = 8) and correlated to mTOR-inhibitor everolimus response., Results: High p-mTOR expression correlated significantly with worse OS in multivariate analysis in the whole patient cohort [Hazar Ratio (HR) 1.06, 95%CI 1.01-1.11, p = 0.03] and in the cisplatin/carboplatin group with both worse OS (HR 1.09, 95%CI 1.02-1.16, p = 0.02) and DFS (HR 1.06, 95%CI 1.00-1.12, p = 0,04). p-ERK expression correlated significantly with DFS in univariate analysis in the whole patient cohort (HR 1.03, 95%CI 1.00-1.05, p = 0.04) and cisplatin/carboplatin group (HR 1.03, 95%CI 1.00-1.07, p = 0.04). PTEN-expression did not correlate with OS/DFS/LRC. Better organoid response to everolimus correlated significantly to higher p-mTOR expression (Rs = - 0.731, p = 0.04)., Conclusions: High p-mTOR expression predicts and high p-ERK expression tends to predict worse treatment outcome in HPV negative HNSCC patients treated with chemoradiation, providing additional evidence that these markers are candidate prognostic biomarkers for survival in this patient population. Also this study shows that the use of HNSCC organoids for biomarker research has potential. The role of PTEN expression as prognostic biomarker remains unclear, as consistent evidence on its prognostic and predictive value is lacking., (© 2023. The Author(s).)

Head and neck squamous cell carcinoma (HNSCC) is an immunogenic cancer type, and tumor associated macrophages (TAMs) are a major component of the tumor microenvironment (TME). In this systematic review and meta-analysis, studies assessing tumor infiltration with CD68+, iNOS+, HLA-DR+, CD11b+, CD163+, CD206+, and CD204+TAMs were included, and correlation to survival hazard was studied. A low number of CD68+TAMs correlated to better overall survival (OS) in multivariate analysis (HR 1.36 95 %CI (1.07-1.72) P = .01). CD68+TAMs did not correlate to disease free survival (DFS), disease specific survival (DSS), progression free survival (PFS), or recurrence free survival (RFS). A low number of CD163+TAMs correlated to better OS in uni- and multivariate analysis (resp. HR 2.65 95 %CI (1.57-4.46) P = .01 and HR 2.42 95 %CI (1.72-3.41) P < .001). A low number of CD163+TAMs also correlated to better DFS and PFS, whereas a low number of CD204+TAMs only correlated to PFS. While IHC analysis of pan macrophage marker CD68 and M2-like marker CD163 both show prognostic utility in OS, CD163 is a stronger prognosticator, as indicated by multivariate meta-analysis. CD163+TAMs also correlate to DFS and PFS; outcomes that are more relevant to patients, thus showing promising results for future clinical implementation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Definitive concomitant cisplatin-based chemoradiotherapy (CRT) is the current gold standard for most patients with advanced stage head and neck squamous cell carcinoma (HNSCC) of the pharynx and larynx. Since previous meta-analysis on CRT outcomes in HNSCC have been reported, advances have been made in radiotherapy techniques and clinical management, while HPV-status has been identified as a strong confounding prognostic factor in oropharyngeal cancer. Here, we present real-world outcome data from a large multicenter cohort of HPV-negative advanced stage HNSCC treated with CRT using contemporary IMRT-based techniques., Method: Retrospective data were collected from a multicenter cohort of 513 patients treated with definitive concurrent platinum-based CRT with curative intent between January 2009 and August 2017. Only patients with HPV-negative advanced stage (III-IV) HNSCC were included. A prognostic model for outcome was developed based on clinical parameters and compared to TNM., Results: Nearly half of the 513 patients (49%) had an oropharyngeal tumor, often locally advanced (73.3% T3-T4b) and with involvement of the regional lymph nodes (84%). Most patients (84%) received cisplatin as single agent. In total 66% received the planned number of cycles and 75% reached a cumulative cisplatin dose of ≥200 mg/m 2 . Locoregional control was achieved in 324 (63%) patients during follow-up, and no association with tumor sites was observed (p = 0.48). Overall survival at 5 year follow-up was 47%, with a better survival for laryngeal cancer (p = 0.02) compared to other sites. A model with clinical variables (gender, high pre-treatment weight loss, N2c/N3-stage and <200 mg/m 2 dose of cisplatin) provided a noticeably stronger association with overall survival than TNM-staging (C- index 0.68 vs 0.55). Simultaneous Integrated Boosting (SIB) significantly outperformed Sequential Boosting (SEQ) to reduce the development of distant metastasis (SEQ vs SIB: OR 1.91 (1.11-3.26; p = 0.02)., Conclusion: Despite advances in clinical management, more than a third of patients with HPV-negative HNSCC do not complete CRT treatment protocols due to cisplatin toxicity. A model that consists of clinical variables and treatment parameters including cisplatin dose provided the strongest association with overall survival. Since cisplatin toxicity is a major obstacle in completing definitive CRT, the development of alternative and less toxic radiosensitizers is therefore warranted to improve treatment results. The association of RT-boost technique with distant metastasis is an important finding and requires further study., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

This study aimed to assess the prognostic value of intratumoral CD57+ cells in head and neck squamous cell carcinoma (HNSCC) and to examine the reproducibility of these analyses using QuPath. Pretreatment biopsies of 159 patients with HPV-negative, stage III/IV HNSCC treated with chemoradiotherapy were immunohistochemically stained for CD57. The number of CD57+ cells per mm 2 tumor epithelium was quantified by two independent observers and by QuPath, software for digital pathology image analysis. Concordance between the observers and QuPath was assessed by intraclass correlation coefficients (ICC). The correlation between CD57 and clinicopathological characteristics was assessed; associations with clinical outcome were estimated using Cox proportional hazard analysis and visualized using Kaplan-Meier curves. The patient cohort had a 3-year OS of 65.8% with a median follow-up of 54 months. The number of CD57+ cells/mm 2 tumor tissue did not correlate to OS, DFS, or LRC. N stage predicted prognosis (OS: HR 0.43, p = 0.008; DFS: HR 0.41, p = 0.003; LRC: HR 0.24, p = 0.007), as did WHO performance state (OS: HR 0.48, p = 0.028; LRC: 0.33, p = 0.039). Quantification by QuPath showed moderate to good concordance with two human observers (ICCs 0.836, CI 0.805-0.863, and 0.741, CI 0.692-0.783, respectively). In conclusion, the presence of CD57+ TILs did not correlate to prognosis in advanced stage, HPV-negative HNSCC patients treated with chemoradiotherapy. Substantial concordance between human observers and QuPath was found, confirming a promising future role for digital, algorithm driven image analysis., (© 2022. The Author(s).)

Aims: Programmed death-ligand 1 (PD-L1) immunostaining is used to predict which non-small-cell lung cancer (NSCLC) patients will respond best to treatment with programmed cell death protein 1/PD-L1 inhibitors. PD-L1 immunostaining is sometimes performed on alcohol-fixed cytological specimens instead of on formalin-fixed paraffin-embedded (FFPE) biopsies or resections. We studied whether ethanol prefixation of clots from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) results in diminished PD-L1 immunostaining as compared with formalin fixation., Methods and Results: FFPE cell blocks from EBUS-TBNA specimens of 54 NSCLC patients were identified. For each case, paired samples were available, consisting of clots directly immersed in formalin and clots prefixed in Fixcyt (50% ethanol). Serial sections were immunostained for PD-L1 by use of the standardised SP263 assay and the 22C3 antibody as a laboratory-developed test (LDT). PD-L1 positivity was determined with two cut-offs (1% and 50%). Concordance of PD-L1 positivity between the formalin-fixed (gold standard) and ethanol-prefixed material was assessed. When the 22C3 LDT was used, 30% and 36% of the ethanol-prefixed specimens showed false-negative results at the 1% and 50% cut-offs, respectively (kappa 0.64 and 0.68). When SP263 was used, 22% of the ethanol-prefixed specimens showed false-negative results at the 1% cut-off (kappa 0.67). At the 50% cut-off, concordance was higher (kappa 0.91), with 12% of the ethanol-prefixed specimens showing false-negative results., Conclusion: Ethanol fixation of EBUS-TBNA specimens prior to formalin fixation can result in a considerable number of false-negative PD-L1 immunostaining results when a 1% cut-off is used and immunostaining is performed with SP263 or the 22C3 LDT. The same applies to use of the 50% cut-off when immunostaining is performed with the 22C3 LDT., (© 2021 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Collagens are a primary component of the extracellular matrix and are functional ligands for the inhibitory immune receptor leukocyte-associated immunoglobulin-like receptor (LAIR)-1. LAIR-2 is a secreted protein that can act as a decoy receptor by binding collagen with higher affinity than LAIR-1. We propose that collagens promote immune evasion by interacting with LAIR-1 expressed on immune cells, and that LAIR-2 releases LAIR-1-mediated immune suppression. Analysis of public human datasets shows that collagens, LAIR-1 and LAIR-2 have unique and overlapping associations with survival in certain tumors. We designed a dimeric LAIR-2 with a functional IgG1 Fc tail, NC410, and showed that NC410 increases human T cell expansion and effector function in vivo in a mouse xenogeneic-graft versus-host disease model. In humanized mouse tumor models, NC410 reduces tumor growth that is dependent on T cells. Immunohistochemical analysis of human tumors shows that NC410 binds to collagen-rich areas where LAIR-1 + immune cells are localized. Our findings show that NC410 might be a novel strategy for cancer immunotherapy for immune-excluded tumors., Competing Interests: MR, Ed, CS, AP, AS, EE, SV, JS, JB, ZC, CJ, NW, MK, LL, SL, SW, DF, LM No competing interests declared, LT LT, CS, AP, JS, JB, ZC, LL, SL and DF are employees from Nextcure. Nextcure holds a patent on NC410. (PCT/US20 17/0453 10)., (© 2021, Ramos et al.)

Expression of programmed cell death-ligand 1 (PD-L1) is being used as predictive biomarker for immunotherapy in head and neck squamous cell carcinoma (HNSCC). Several antibodies are available for PD-L1 testing and multiple staining and scoring methods are used. This study aimed to compare the performance of two PD-L1 standardized assays (SP263 and 22C3 pharmDx) and one laboratory-developed test (LDT) (22C3) in HNSCC using the tumor proportion score (TPS) and the combined positive score (CPS). Pretreatment biopsies from 147 HNSCC patients were collected in a tissue-microarray (TMA). Serial sections of the TMA were immunohistochemically stained for PD-L1 expression using 22C3 pharmDx on the Dako Link 48 platform, SP263 on the Ventana Benchmark Ultra platform, and 22C3 as an LDT on the Ventana Benchmark Ultra. Stained slides were assessed for TPS and CPS. Cutoffs of ≥1% and ≥50% for TPS and ≥1 and ≥20 for CPS were used. Concordance between the different staining assays was moderate to poor for TPS (intraclass correlation coefficient (ICC) 0.46) as well as for CPS (ICC 0.34). When stratifying patients by clinically relevant cutoffs, considerable differences between the assays were observed: concordance was poor for both TPS and CPS. Generally, SP263 stained a higher percentage of cells than the other assays, especially when using the CPS. Moderate concordance was shown between three different PD-L1 immunohistochemical assays and considerable differences in PD-L1 positivity were observed when using clinically relevant cutoffs. This should be taken into account when using PD-L1 expression to guide clinical practice.

Histologic grade is a biomarker that is widely used to guide treatment of invasive breast cancer (IBC) and ductal carcinoma in situ of the breast (DCIS). Yet, currently, substantial grading variation between laboratories and pathologists exists in daily pathology practice. This study was conducted to evaluate whether an e-learning may be a feasible tool to decrease grading variation of (pre)malignant breast lesions. An e-learning module, representing the key-concepts of grading (pre)malignant breast lesions through gold standard digital images, was designed. Pathologists and residents could take part in either or both the separate modules on DCIS and IBC. Variation in grading of a digital set of lesions before and after the e-learning was compared in a fully-crossed study-design. Multiple outcome measures were assessed: inter-rater reliability (IRR) by Light's kappa, the number of images graded unanimously, the number of images with both extreme scores (i.e., grade I and grade III), and the average number of discrepancies from expert-consensus. Participants were included as they completed both the pre- and post-e-learning set (DCIS-module: n = 36, IBC-module: n = 21). For DCIS, all outcome measures improved after e-learning, with the IRR improving from fair (kappa: 0.532) to good (kappa: 0.657). For IBC, all outcome measures for the subcategories tubular differentiation and mitosis improved, with >90% of participants agreeing on almost 90% of the images after the e-learning. In contrast, the IRR for the subcategory of nuclear pleomorphism remained fair (kappa: 0.523 vs. kappa: 0.571). This study shows that an e-learning module, in which pathologists and residents are trained in histologic grading of DCIS and IBC, is a feasible and promising tool to decrease grading variation of (pre)malignant breast lesions. This is highly relevant given the important role of histologic grading in clinical decision making of (pre)malignant breast lesions.

Background : Despite the improvement in therapeutic interventions, 5-year survival rates in Head and Neck Squamous Cell Carcinoma (HNSCC) are limited. HNSCC is an immunogenic cancer type for which molecular stratification markers are lacking. Tumor-infiltrating lymphocytes (TILs) have shown a favorable prognostic role in different cancer types. This study focused on the prognostic role of NK cells in HNSCC. Methods : A systematic search was conducted in Pubmed/Medline and Embase. Articles that correlated the presence of intratumoral NK cells, activating/inhibiting receptors, death receptors, or their ligands with clinicopathologic characteristics or survival were included. A meta-analysis was performed that assessed the association between CD56+ and CD57+ and overall survival (OS), disease-free survival (DFS), and progression-free survival (PFS). Results : A pooled analysis indicated a favorable prognostic role of CD56+ and CD57+ NK cells for OS (HR 0.19 CI 0.11-0.35). NK cell markers NKp46 and Granzyme B (GrB) also have a favorable prognostic role. NK cell ligand Fas correlated with better survival and better characteristics. NK cell marker Fas-L, NK cell ligands CEACAM1, RCAS1, CD70 and TRAIL-R, and effector molecules of these ligands, FADD and FAP1, correlated to features of worse prognosis. Conclusion : A favorable prognostic role of NK cells in HNSCC was found in this review. Some studies implied the opposite, indicating the fine balance between pro- and anti-tumor functions of NK cells. Future studies using homogeneous patient cohorts regarding tumor subsite and treatment modality, are necessary to further provide insight into the prognostic role of NK cells., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Aim: This study aimed to evaluate the presence and prognostic value of tumor-infiltrating T cells in the tumor epithelium in advanced stage, HPV-negative head and neck squamous cell carcinoma (HNSCC) patients treated with primary chemoradiotherapy using digital pathology., Methods: Pre-treatment biopsies from 80 oropharyngeal, 52 hypopharyngeal, and 29 laryngeal cancer patients were collected in a tissue microarray (TMA) and immunohistochemically stained for T-cell markers CD3, CD4, CD8, FoxP3, and PD1, and for immune checkpoint PD-L1. For each marker, the number of positive tumor-infiltrating lymphocytes (TILs) per mm 2 tumor epithelium was digitally quantified and correlated to overall survival (OS), disease-free survival (DFS), and locoregional control (LRC), as well as to clinicopathological characteristics. Differences in clinical outcome were estimated using Cox proportional hazard analysis and visualized using Kaplan-Meier curves., Results: The patient cohort had a 3-year OS of 58%, with a median follow-up of 53 months. None of the T-cell markers showed a correlation with OS, DFS or LRC. A low N stage was correlated to a better prognosis (OS: HR 0.39, p = 0.0028, DFS: HR 0.34, p =  < 0.001, LRC: HR 0.24, p = 0.008). High TIL counts were more often observed in PD-L1-positive tumors (p < 0.05)., Conclusion: This study showed an objective, digital pathology-aided method to assess TILs in the tumor epithelium. However, it did not provide evidence for a prognostic role of the presence of CD3 + , CD4 + , CD8 + , FoxP3 + , and PD1 + TILs in the tumor epithelium of advanced stage, HPV-negative HNSCC patients treated with primary chemoradiotherapy.

Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)-derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro . Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs. SIGNIFICANCE: This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice. See related commentary by Hill and D'Andrea, p. 828 . This article is highlighted in the In This Issue feature, p. 813 ., (©2019 American Association for Cancer Research.)

Background - The presence of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment is associated with an improved prognosis and a better response to therapy in different types of cancer. In this systematic review and meta-analysis, we investigated the prognostic value of T cells in head and neck squamous cell carcinoma (HNSCC). Methods - In a systematic review, Pubmed and Embase were searched for publications that investigated the prognostic value of T cells in HNSCC. A meta-analysis was performed including all studies assessing the association between CD3+, CD4+, CD8+, and FoxP3+ TILs and overall survival (OS), disease-free survival (DFS), or locoregional control (LRC). Results - A pooled analysis indicated a favorable, prognostic role for CD3+ TILs (HR 0.64 (95%CI 0.47-0.85) for OS, HR 0.63 (95%CI 0.49-0.82) for DFS) and CD8+ TILs (HR 0.67 (95%CI 0.58-0.79) for OS, HR 0.50 (95%CI 0.37-0.68) for DFS, and HR 0.82 (95%CI 0.70-0.96) for LRC) in the clinical outcome of HNSCC. FoxP3+ TILs were also associated with better OS (HR 0.80 (95%CI 0.70-0.92)). Conclusion - This systematic review and meta-analysis confirmed the favorable, prognostic role of CD3+ and CD8+ T cell infiltration in HNSCC patients and found an association between FoxP3+ TILs and improved overall survival. Future studies using homogeneous patient cohorts with regard to tumor subsite, stage and treatment are necessary to provide more insight in the predictive value of TILs in HNSCC.

Competing Interests: The authors have no conflicts of interest to declare.