NEMO/NF-κB signaling functions as a double-edged sword in PanIN formation versus progression to pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is marked by a dismal survival rate, lacking effective therapeutics due to its aggressive growth, late-stage diagnosis, and chemotherapy resistance. Despite debates on NF-κB targeting for PDAC treatment, no successful approach has emerged. METHODS: To elucidate the role of NF-κB, we ablated NF-κB essential modulator (NEMO), critical for conventional NF-κB signaling, in the pancreata of mice that develop precancerous lesions (KC mouse model). Secretagogue-induced pancreatitis by cerulein injections was utilized to promote inflammation and accelerate PDAC development. RESULTS: NEMO deletion reduced fibrosis and inflammation in young KC mice, resulting in fewer pancreatic intraepithelial neoplasias (PanINs) at later stages. Paradoxically, however, NEMO deletion accelerated the progression of these fewer PanINs to PDAC and reduced median lifespan. Further, analysis of tissue microarrays from human PDAC sections highlighted the correlation between reduced NEMO expression in neoplastic cells and poorer prognosis, supporting our observation in mice. Mechanistically, NEMO deletion impeded oncogene-induced senescence (OIS), which is normally active in low-grade PanINs. This blockage resulted in fewer senescence-associated secretory phenotype (SASP) factors, reducing inflammation. However, blocked OIS fostered replication stress and DNA damage accumulation which accelerated PanIN progression to PDAC. Finally, treatment with the DNA damage-inducing reagent etoposide resulted in elevated cell death in NEMO-ablated PDAC cells compared to their NEMO-competent counterparts, indicative of a synthetic lethality paradigm. CONCLUSIONS: NEMO exhibited both oncogenic and tumor-suppressive properties during PDAC development. Caution is suggested in therapeutic interventions targeting NF-κB, which may be detrimental during PanIN progression but beneficial post-PDAC development.

Endoscopic Treatment of T1 Colorectal Cancer.

Commonly accepted criteria for curative resection of T1 colorectal cancer include R0 resection with horizontal and vertical clear margins (R0), absence of lympho-vascular or vessel infiltration (L0, V0), a low to moderate histological grading (G1/2), low tumor cell budding, and limited (<1000 µm) infiltration into the submucosa. However, submucosal infiltration depth in the absence of other high-risk features has recently been questioned as a high-risk situation for lymph-node metastasis. Consequently, endoscopic resection techniques should focus on the acquisition of qualitatively and quantitively sufficient submucosal tissue. Here, we summarize the current literature on lymph-node metastasis risk after endoscopic resection of T1 colorectal cancer. Moreover, we discuss different endoscopic resection techniques with respect to the quality of the resected specimen.

Slide-to-Slide Tissue Transfer and Array Assembly From Limited Samples for Comprehensive Molecular Profiling.

Tissue microarrays (TMA) have become an important tool in high-throughput molecular profiling of tissue samples in the translational research setting. Unfortunately, high-throughput profiling in small biopsy specimens or rare tumor samples (eg, orphan diseases or unusual tumors) is often precluded owing to limited amounts of tissue. To overcome these challenges, we devised a method that allows tissue transfer and construction of TMAs from individual 2- to 5-µm sections for subsequent molecular profiling. We named the technique slide-to-slide (STS) transfer, and it requires a series of chemical exposures (so-called xylene-methacrylate exchange) in combination with rehydrated lifting, microdissection of donor tissues into multiple small tissue fragments (methacrylate-tissue tiles), and subsequent remounting on separate recipient slides (STS array slide). We developed the STS technique by assessing the efficacy and analytical performance using the following key metrics: (a) dropout rate, (b) transfer efficacy, (c) success rates using different antigen-retrieval methods, (d) success rates of immunohistochemical stains, (e) fluorescent in situ hybridization success rates, and (f) DNA and (g) RNA extraction yields from single slides, which all functioned appropriately. The dropout rate ranged from 0.7% to 6.2%; however, we applied the same STS technique successfully to fill these dropouts ("rescue" transfer). Hematoxylin and eosin assessment of donor slides confirmed a transfer efficacy of >93%, depending on the size of the tissue (range, 76%-100%). Fluorescent in situ hybridization success rates and nucleic acid yields were comparable with those of traditional workflows. In this study, we present a quick, reliable, and cost-effective method that offers the key advantages of TMAs and other molecular techniques-even when tissue is sparse. The perspectives of this technology in biomedical sciences and clinical practice are promising, given that it allows laboratories to create more data with less tissue.

BTK Isoforms p80 and p65 Are Expressed in Head and Neck Squamous Cell Carcinoma (HNSCC) and Involved in Tumor Progression.

Here, we describe the expression of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.

Diagnostic quality model (DQM): an integrated framework for the assessment of diagnostic quality when using AI/ML.

BACKGROUND: Laboratory medicine has reached the era where promises of artificial intelligence and machine learning (AI/ML) seem palpable. Currently, the primary responsibility for risk-benefit assessment in clinical practice resides with the medical director. Unfortunately, there is no tool or concept that enables diagnostic quality assessment for the various potential AI/ML applications. Specifically, we noted that an operational definition of laboratory diagnostic quality - for the specific purpose of assessing AI/ML improvements - is currently missing. METHODS: A session at the 3rd Strategic Conference of the European Federation of Laboratory Medicine in 2022 on "AI in the Laboratory of the Future" prompted an expert roundtable discussion. Here we present a conceptual diagnostic quality framework for the specific purpose of assessing AI/ML implementations. RESULTS: The presented framework is termed diagnostic quality model (DQM) and distinguishes AI/ML improvements at the test, procedure, laboratory, or healthcare ecosystem level. The operational definition illustrates the nested relationship among these levels. The model can help to define relevant objectives for implementation and how levels come together to form coherent diagnostics. The affected levels are referred to as scope and we provide a rubric to quantify AI/ML improvements while complying with existing, mandated regulatory standards. We present 4 relevant clinical scenarios including multi-modal diagnostics and compare the model to existing quality management systems. CONCLUSIONS: A diagnostic quality model is essential to navigate the complexities of clinical AI/ML implementations. The presented diagnostic quality framework can help to specify and communicate the key implications of AI/ML solutions in laboratory diagnostics.

Targetable alterations in primary extranodal diffuse large B-cell lymphoma.

Primary extranodal diffuse large B-cell lymphoma (PE-DLBCL) is a heterogeneous subgroup of DLBCL. We investigated the prevalence and prognostic value of surface expression of PD-L1, PD1, and CD30, copy number of 9p24.1 (PD-L1 region), and mutations in MYD88, CD79B, CARD11, and BTK in a cohort of 116 patients, localized in the mediastinum (PMBL, n = 12), ear, nose and throat (ENT, n = 28), central nervous system (n = 29), testis (n = 7), breast (n = 4), stomach (n = 10), bone (n = 8), spleen (n = 2), and skin (n = 16). PD-L1 expression is most frequent in PMBL (92%), followed by lymphomas originating in the stomach (57%), ENT (23%), and skin (18%). PD1 was expressed at low levels in less than 13% of PE-DLBCL, while CD30 expression was found in 58% of PMBL. Mutation analysis revealed an unexpectedly high frequency of MYD88 and CD79B mutations in ENT lymphomas (46% and 50%, respectively). CARD11 mutations are rare but more frequently found in gastric lymphomas (30%), suggesting BTK resistance. Thirty-four of 113 (30%) of the lymphomas harbored both MYD88 and CD79B mutations. Lower overall and progression-free survival rates were found for cases with MYD88, CD79B, and BTK mutations. These data confirm the biologic singularity of PE-DLBCLs and provide some suggestions for targeted therapies.

Changes in Gene Expression Patterns in the Tumor Microenvironment of Head and Neck Squamous Cell Carcinoma Under Chemoradiotherapy Depend on Response.

Chemoradiotherapy (CRT) is a standard treatment for advanced head and neck squamous cell carcinoma (HNSCC). Unfortunately, not all patients respond to this therapy and require further treatment, either salvage surgery or palliative therapy. The addition of immunotherapy to CRT is currently being investigated and early results describe a mixed response. Therefore, it is important to understand the impact of CRT on the tumor microenvironment (TME) to be able to interpret the results of the clinical trials. Paired biopsies from 30 HNSCC patients were collected before and three months after completion of primary CRT and interrogated for the expression of 1392 immune- and cancer-related genes. There was a relevant difference in the number of differentially expressed genes between the total cohort and patients with residual disease. Genes involved in T cell activation showed significantly reduced expression in these tumors after therapy. Furthermore, gene enrichment for several T cell subsets confirmed this observation. The analysis of tissue resident memory T cells (TRM) did not show a clear association with impaired response to therapy. CRT seems to lead to a loss of T cells in patients with incomplete response that needs to be reversed. It is not clear whether the addition of anti-PD-1 antibodies alone to CRT can prevent treatment failure, as no upregulation of the targets was measurable in the TME.

Patterns of Tumor Infiltrating Lymphocytes in Adenoid Cystic Carcinoma of the Head and Neck.

Adenoid cystic carcinoma (ACC) is a rare malignancy in the head and neck. The prognosis remains poor and late recurrences often occur after 5 years and later. To date, there are no reliable prognostic markers for ACC. In several solid tumors, tertiary lymphoid structures (TLS) are associated with improved survival. This study aims to investigate the role of distribution patterns of tumor infiltrating immune cells (TIL) in ACC. A cohort of 50 patients from three different cancer centers was available for analysis. Sections were stained for CD3, CD4, CD8 and CD20 and evaluated with regard to their distribution of TIL. Patterns were determined as infiltrated-excluded, infiltrated-inflamed and presence of tertiary lymphoid structures. About half of the cases showed an infiltrated-excluded TIL pattern and only a minority of six cases had TLS present within the tumor. Within the inflamed phenotype CD3+ cells were by far the most abundant lymphocyte subtype, and within this compartment, CD8+ T cells were predominant. There was no influence on overall or disease-free survival by any of the TIL patterns. This indicates that ACC is a tumor with very low immunogenicity and even abundance of lymphocytes does not seem to improve prognosis for this disease. Therefore, the observed lack of response towards immunotherapy is not surprising and other methods to induce recognition of ACC by the immune system must be found.

Combination of two monoclonal antibodies with SOCS1 N- and C-terminal binding sites to address SOCS1 status in B cells and B-cell lymphoma.

INTRODUCTION: Accumulating studies show that the tumour suppressor SOCS1 is one of the most frequently mutated genes in lymphomas, often affecting the coding sequence of SOCS1 protein. Depending on the type of mutation and lymphoma concerned, SOCS1 mutations have different impacts on progression-free and overall survival. Two antibodies binding the N and C terminals of SOCS1 would be a suitable 'test pair' to identify truncated versions of SOCS1. We, therefore, compared the C-terminal antibody 424C with the N-terminal antibody 4H1. MATERIALS AND METHODS: As 424C has already been characterised, we performed a comparative analysis of anti-SOCS1 antibody 4H1 using immunohistochemistry on human tonsil tissue and chamber slides, immunoblots on SOCS1 wildtype and mutated transfected HEK293T cells and lymphoma cell lines and cross-reactivity analysis and epitope mapping with protein microarrays. RESULTS: Compared with 424C, anti-SOCS1 antibody 4H1 showed various cross-reactions with other proteins resulting in a 'pancellular' immunohistochemical staining pattern in FFPE lymphoid tissue. Like 424C, 4H1 identified SOCS1 wildtype and SOCS1 mutations in immunoblot experiments but also bound an unknown protein with high intensity. CONCLUSION: Anti-SOCS1 antibody 4H1 may be useful in a molecular setting but is disqualified as an immunohistochemical diagnostic tool due to its very broad non-specific binding.

Single-cell-resolved differentiation of human induced pluripotent stem cells into pancreatic duct-like organoids on a microwell chip.

Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facilitates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pancreatic progenitors through ductal intermediates to two types of mature duct-like cells and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial-mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models.

A new reliable and highly specific monoclonal antibody to detect the C-terminal region of silencer of cytokine signaling 1.

INTRODUCTION: SOCS1, a negative regulator of JAK/STAT signaling, is among the most frequently mutated genes in DLBCL and classical Hodgkin lymphoma. The C-terminal SOCS box domain, mediating the degradation of phospho-JAK2, is often affected or even lacking. The analysis of such variants is hampered by the lack of a SOCS1-specific monoclonal antibody recognizing the C-terminus of SOCS1. As this C-terminus is often lost or mutated in B-cell lymphomas, staining with amino-terminal targeting antibodies in a lymphoma setting might be misleading. METHODS: BALB/c mice were immunized with a truncated SOCS1 C-terminal protein. The supernatant of generated hybridoma cells was screened by ELISA and, immunohistochemically, on formalin-fixed and paraffin-embedded tonsil. After antibody purification by affinity chromatography, epitope mapping and cross-reactivity check followed via substitution scans. SOCS1 protein expression was investigated on cell cultures and cytoblocks of SOCS1WT stably transfected HEK293T cells, lymphoma cell lines and lymphoid tissues. RESULTS: Procedures resulted in one monoclonal IgG1 anti-SOCS1 antibody, 424C, that recognizes and strongly binds to the C-terminal region of SOCS1 in immunoblot and immunohistochemistry analyses. CONCLUSION: This new anti-SOCS1 monoclonal antibody is a valuable tool to detect SOCS1 expression dependent on an existing SOCS1 box and, therefore, indicating a full-length SOCS1 protein.

RINT1 Regulates SUMOylation and the DNA Damage Response to Preserve Cellular Homeostasis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) still presents with a dismal prognosis despite intense research. Better understanding of cellular homeostasis could identify druggable targets to improve therapy. Here we propose RAD50-interacting protein 1 (RINT1) as an essential mediator of cellular homeostasis in PDAC. In a cohort of resected PDAC, low RINT1 protein expression correlated significantly with better survival. Accordingly, RINT1 depletion caused severe growth defects in vitro associated with accumulation of DNA double-strand breaks (DSB), G2 cell cycle arrest, disruption of Golgi-endoplasmic reticulum homeostasis, and cell death. Time-resolved transcriptomics corroborated by quantitative proteome and interactome analyses pointed toward defective SUMOylation after RINT1 loss, impairing nucleocytoplasmic transport and DSB response. Subcutaneous xenografts confirmed tumor response by RINT1 depletion, also resulting in a survival benefit when transferred to an orthotopic model. Primary human PDAC organoids licensed RINT1 relevance for cell viability. Taken together, our data indicate that RINT1 loss affects PDAC cell fate by disturbing SUMOylation pathways. Therefore, a RINT1 interference strategy may represent a new putative therapeutic approach. SIGNIFICANCE: These findings provide new insights into the aggressive behavior of PDAC, showing that RINT1 directly correlates with survival in patients with PDAC by disturbing the SUMOylation process, a crucial modification in carcinogenesis.

[Primary small cell neuroendocrine carcinoma of the larynx: a review of literature and case series]. / Kleinzelliges neuroendokrines Karzinom des Kopf-Hals-Bereichs: eine Übersichtsarbeit und Fallserie.

INTRODUCTION: Small cell neuroendocrine carcinoma (SCNC) of the larynx is a rare tumor entity with a 5-year overall survival (OS) of only 5 % after treatment with chemoradiotherapy. METHODS: A systematic review of the literature was performed for "SCNC" and "SCNC in head and neck". Our hospital's own electronic patient file database was investigated for patients diagnosed with a SCNC over the last 12 years. RESULTS: The effectiveness of chemoradiotherapy in SCNC is still unclear since randomized clinical trials are missing for the evaluation of standard of care treatment. Common therapy approaches are based on experiences with small cell lung cancer. 0.5 % of all SCNC occur in the head and neck region. In the last 12 years, we diagnosed 9 patients with SCNC, two of which were located in the larynx. Exemplarily, we report the case of a 29-year-old male with the initial diagnosis of a SCNC of the larynx with concurrent lymph node metastasis. This case is particularly interesting due to the young age at disease onset and the lack of major risk factors. Treatment was modified to nivolumab due to progressive disease after treatment with chemoradiotherapy. After an OS of 22 months, the patient deceased due to a tumor-associated major bleeding with airway obstruction. CONCLUSION: So far there are no clinical reports evaluating the use of nivolumab in third-line-therapy of SCNC. NTRK fusion (neurotrophic tyrosine receptor kinase gene fusion) or the folate receptor expression analysis should be considered to evaluate the potential use of a tropomyosin receptor kinase inhibitor or a folate receptor targeting therapy.

Immune checkpoint expression in HNSCC patients before and after definitive chemoradiotherapy.

BACKGROUND: Primary platinum-based chemoradiotherapy (CRT) remains the treatment of choice for nonresectable squamous cell carcinoma of the head and neck (HNSCC). Immune-checkpoint modulators are used as palliative therapy and studied in combination with definitive CRT. However, the immunological changes by CRT need yet to be understood. METHODS: A cohort consisting of 67 paired tissue biopsies (N = 134) of HNSCC patients before and after CRT was created. The expression of PD-1, PD-L1, and CD27 of tumor and immune cells by immunohistochemistry was evaluated. RESULTS: PD-L1 expression on immune cells of non-responders was significantly lower before CRT (P = .008). CD27 was expressed only on immune cells and not on cancer cells. A significant lower CD27-expression score was observed following CRT (P = .019). CONCLUSIONS: Conventional CRT changes the expression of CD27 in the tumor microenvironment. Whether this is due to a loss of expression or a reduction of CD27+ cells must be evaluated in further analyses.

Immune Checkpoint Expression on Immune Cells of HNSCC Patients and Modulation by Chemo- and Immunotherapy.

Endogenous control mechanisms, including immune checkpoints and immunosuppressive cells, are exploited in the process of tumorigenesis to weaken the anti-tumor immune response. Cancer treatment by chemotherapy or immune checkpoint inhibition can lead to changes of checkpoint expression, which influences therapy success. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were isolated from head and neck squamous cell carcinoma (HNSCC) patients (n = 23) and compared to healthy donors (n = 23). Immune checkpoint expression (programmed cell death ligand 1 (PD-1), tumor necrosis factor receptor (TNFR)-related (GITR), CD137, tumor necrosis factor receptor superfamily member 4 (TNFRSF4) (OX40), t-cell immunoglobulin and mucin-domain containing-3 (TIM3), B- and T-lymphocyte attenuator (BTLA), lymphocyte-activation gene 3 (LAG3)) was determined on immune cells by flow cytometry. PD-L1 expression was detected on tumor tissue by immunohistochemistry. Immune cells were treated with immuno- and chemotherapeutics to investigate treatment-specific change in immune checkpoint expression, in vitro. Specific changes of immune checkpoint expression were identified on PBL and TIL of HNSCC patients compared to healthy donors. Various chemotherapeutics acted differently on the expression of immune checkpoints. Changes of checkpoint expression were significantly less pronounced on regulatory T cells compared to other lymphocyte populations. Nivolumab treatment significantly reduced the receptor PD-1 on all analyzed T cell populations, in vitro. The specific immune checkpoint expression patterns in HNSCC patients and the investigated effects of immunomodulatory agents may improve the development and efficacy of targeted immunotherapy.

Physiological levels of the PTEN-PI3K-AKT axis activity are required for maintenance of Burkitt lymphoma.

In addition to oncogenic MYC translocations, Burkitt lymphoma (BL) depends on the germinal centre (GC) dark zone (DZ) B cell survival and proliferation programme, which is characterized by relatively low PI3K-AKT activity. Paradoxically, PI3K-AKT activation facilitates MYC-driven lymphomagenesis in mice, and it has been proposed that PI3K-AKT activation is essential for BL. Here we show that the PI3K-AKT activity in primary BLs and BL cell lines does not exceed that of human non-neoplastic tonsillar GC DZ B cells. BLs were not sensitive to AKT1 knockdown, which induced massive cell death in pAKThigh DLBCL cell lines. Likewise, BL cell lines show low sensitivity to pan-AKT inhibitors. Moreover, hyper-activation of the PI3K-AKT pathway by overexpression of a constitutively active version of AKT (myrAKT) or knockdown of PTEN repressed the growth of BL cell lines. This was associated with increased AKT phosphorylation, NF-κB activation, and downregulation of DZ genes including the proto-oncogene MYB and the DZ marker CXCR4. In contrast to GCB-DLBCL, PTEN overexpression was tolerated by BL cell lines. We conclude that the molecular mechanisms instrumental to guarantee the survival of normal DZ B cells, including the tight regulation of the PTEN-PI3K-AKT axis, also operate in the survival/proliferation of BL.

FOXO1 Confers Maintenance of the Dark Zone Proliferation and Survival Program and Can Be Pharmacologically Targeted in Burkitt Lymphoma.

The FOXO1 transcription factor plays a central role in the proliferation and survival of B cells at several stages of differentiation. B cell malignancies, with exception of classical Hodgkin lymphoma, maintain expression of FOXO1 at levels characteristic for their non-malignant counterparts. Extensive expression profiling had revealed that Burkitt lymphoma (BL) show many characteristics of the dark zone (DZ) germinal center (GC) B cell program. Here we show that FOXO1 knockdown inhibits proliferation of human BL cell lines. The anti-proliferative effect of the FOXO1 knockdown is associated with the repression of the DZ B cell program including expression of MYB, CCND3, RAG2, BACH2, and CXCR4. In addition, the induction of signaling pathways of the light zone (LZ) program like NF-κB and PI3K-AKT was observed. Using a rescue experiment we identified downregulation of the proto-oncogene MYB as a critical factor contributing to the antiproliferative effect of FOXO1 knockdown. In an attempt to estimate the feasibility of pharmacological FOXO1 repression, we found that the small molecular weight FOXO1 inhibitor AS1842856 induces cell death and growth arrest in BL cell lines at low concentrations. Interestingly, we found that overactivation of FOXO1 also induces growth inhibition in BL cell lines, indicating the importance of a tight regulation of FOXO1 activity in BL.

Clinical utility of a protein-based oncopanel in patients with end-stage head and neck cancer.

Aim: In a prospective clinical initiative, we selected heavily pretreated head and neck carcinoma patients and assessed the clinical utility of a protein-based oncopanel for identification of potential targetable markers. Patients & methods: Tumor samples of 45 patients were evaluated using a 12-marker immunohistochemistry panel. The primary end point was the prevalence of potentially actionable markers. Results: At least one expressed marker in each case could be identified. We noted a high prevalence of EGFR (80%, 39/45) and MET (57.4%, 28/45). Three patients received oncopanel-based therapy with variable results. Conclusion: Despite the limited number of treated subjects, oncopanel analysis in end-stage head and neck cancer is operationally and technically feasible. Combination with targeted next generation sequencing might provide additional therapy options.

Somatic mutations and promotor methylation of the ryanodine receptor 2 is a common event in the pathogenesis of head and neck cancer.

Genomic sequencing projects unraveled the mutational landscape of head and neck squamous cell carcinoma (HNSCC) and provided a comprehensive catalog of somatic mutations. However, the limited number of significant cancer-related genes obtained so far only partially explains the biological complexity of HNSCC and hampers the development of novel diagnostic biomarkers and therapeutic targets. We pursued a multiscale omics approach based on whole-exome sequencing, global DNA methylation and gene expression profiling data derived from tumor samples of the HIPO-HNC cohort (n = 87), and confirmed new findings with datasets from The Cancer Genome Atlas (TCGA). Promoter methylation was confirmed by MassARRAY analysis and protein expression was assessed by immunohistochemistry and immunofluorescence staining. We discovered a set of cancer-related genes with frequent somatic mutations and high frequency of promoter methylation. This included the ryanodine receptor 2 (RYR2), which showed variable promoter methylation and expression in both tumor samples and cell lines. Immunohistochemical staining of tissue sections unraveled a gradual loss of RYR2 expression from normal mucosa via dysplastic lesion to invasive cancer and indicated that reduced RYR2 expression in adjacent tissue and precancerous lesions might serve as risk factor for unfavorable prognosis and upcoming malignant conversion. In summary, our data indicate that impaired RYR2 function by either somatic mutation or epigenetic silencing is a common event in HNSCC pathogenesis. Detection of RYR2 expression and/or promoter methylation might enable risk assessment for malignant conversion of dysplastic lesions.

Low SOX2 expression marks a distinct subset of adenoid cystic carcinoma of the head and neck and is associated with an advanced tumor stage.

INTRODUCTION: The transcription factor SOX2 has been identified as a lineage survival oncogene in squamous cell carcinoma and copy number gain is a common event in several human malignancies including head and neck cancer. However, the regulation and function of SOX2 during carcinogenesis as well as its prognostic value appears to be highly context dependent. As an example, high SOX2 expression in lung squamous cell carcinoma (SCC) is related to a favorable prognosis, while it is associated with poor outcome in lung adenocarcinoma. More recently, higher SOX2 levels and improved survival was also reported for head and neck SCC (HNSCC), and silencing of SOX2 expression in HNSCC cell lines revealed a mesenchymal-like phenotype with prominent vimentin expression. So far, SOX2 expression and its clinical relevance for other head and neck cancers, such as adenoid cystic carcinoma (HNACC) have not been sufficiently investigated. MATERIAL AND METHODS: SOX2, vimentin and E-cadherin expression was assessed by immunohistochemical staining on serial sections from formalin fixed and paraffin embedded tissue samples of a patient cohort (n = 45) with primary ACC and correlated with patient and tumor characteristics as well as survival. RESULTS: High SOX2 expression was found in 14 (31%) primary tumor specimens and was significantly correlated with a N0 lymph node status (p = 0.04), while low SOX2 expression was correlated with a solid growth pattern (p = 0.031). Of the 45 patients, 27 tumor samples resembled an EMT-like phenotype, as assessed by high vimentin and low E-cadherin levels. However, in HNACC SOX2 levels were neither correlated with vimentin nor with E-cadherin expression, further supporting a context dependent regulation and function of SOX2 in distinct tumor entities. CONCLUSION: The absence of SOX2 was predominantly found in solid HNACC, which are characterized by a more aggressive phenotype in ACC. However, the underlying molecular mechanisms of SOX2 regulation and function in distinct HNACC subgroups remain to be fully elucidated.