The Gene Expression Landscape of Prostate Cancer BM Reveals Close Interaction with the Bone Microenvironment.

Bone metastatic (BM) prostate cancer (PCa) belongs to the most lethal form of PCa, and therapeutic options are limited. Molecular profiling of metastases contributes to the understanding of mechanisms defining the bone metastatic niche. Our aim was to explore the transcriptional profile of PCa BM and to identify genes that drive progression. Paraffin-embedded tissues of 28 primary PCa and 30 BM were submitted to RNA extraction and analyzed by RNA sequencing using the Nanostring nCounter gene expression platform. A total of 770 cancer-related genes were measured using the Nanostring™ PanCancer progression panel. Gene Ontology (GO), KEGG, Reactome, STRING, Metascape, PANTHER, and Pubmed were used for data integration and gene annotation. We identified 116 differentially expressed genes (DEG) in BM compared to primaries. The most significant DEGs include CD36, FOXC2, CHAD, SPP1, MMPs, IBSP, and PTX3, which are more highly expressed in BM, and ACTG2, MYH11, CNN1, FGF2, SPOCK3, and CHRDL1, which have a lower expression. DEGs functionally relate to extracellular matrix (ECM) proteoglycans, ECM-receptors, cell-substrate adhesion, cell motility as well as receptor tyrosine kinase signaling and response to growth factors. Data integration and gene annotation of 116 DEGs were used to build a gene platform which we termed "Manually Annotated and Curated Nanostring-data Platform". In summary, our results highlight the significance of certain genes in PCa BM to which essential pro-metastatic functions could be ascribed. Data from this study provide a comprehensive platform of genes that are related to PCa BM and provide evidence for further investigations.

[The tumor microenvironment-relay station for prognosis and therapy response]. / Die Tumormikroumgebung ­ Relaisstation für Prognose und Therapieverhalten.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with a dismal prognosis. Besides tobacco and alcohol abuse, human papilloma virus (HPV) infection is an independent risk factor, particularly in oropharyngeal squamous cell carcinomas (OPSCC). One key determinant of therapy response and prognosis is the tumor immune microenvironment (TIME). A strong anti-tumor immune response represents an important mechanism of tumor cell killing. Another major determinant is the tumor metabolism. One feature of tumor cells is their reliance on glycolysis instead of oxidative phosphorylation (OXPHOS) for energy production despite sufficient oxygen supply.The presented studies were based on several different clinically and pathologically well annotated HNSCC cohorts. Immunohistochemical stainings were performed on tissue microarrays and whole slides against p16, different immune cell markers, and metabolic markers. DNA and mRNA were extracted to detect HPV and assess immune-related and metabolic genes via RT-PCR and Nanostring.An initial assessment of HPV infection frequencies in different HNSCC cohorts showed that socioeconomic and regional factors strongly influence HPV prevalence. An analysis of the immunological and metabolic differences of HPV-positive and HPV-negative OPSCC demonstrated an enhanced anti-tumor immune response together with increased levels of OXPHOS metabolism in HPV-positive OPSCC. In a subsequent study, a strong anti-tumor immune response together with an OXPHOS metabolism was associated with improved short-term survival in HNSCC. Finally, TIME differences of HNSCC primary tumors and recurrent tumors were analyzed to understand the poor therapeutic response of recurrences and discovered an overall decrease of tumor infiltrating lymphocytes with significant loss of CD8-positive T cells and B lymphocytes as well as a significant decrease of tertiary lymphoid structures in recurrences after chemoradiation.These results demonstrate the clinical and molecular differences of HPV-positive and HPV-negative HNSCC with a focus on OPSCC. Furthermore, they delineate the interdependencies of TIME and tumor metabolism in HNSCC in general and stress their impact on therapy response. Additionally, they show an impairment of anti-tumor immune response in recurrences after chemoradiation, which indicates immune evasion as one recurrence driver.

Mapping of ICD-O Tuples to OncoTree Codes Using SNOMED CT Post-Coordination.

Around 500,000 oncological diseases are diagnosed in Germany every year which are documented using the International Classification of Diseases for Oncology (ICD-O). Apart from this, another classification for oncology, OncoTree, is often used for the integration of new research findings in oncology. For this purpose, a semi-automatic mapping of ICD-O tuples to OncoTree codes was developed. The implementation uses a FHIR terminology server, pre-coordinated or post-coordinated SNOMED CT expressions, and subsumption testing. Various validations have been applied. The results were compared with reference data of scientific papers and manually evaluated by a senior pathologist, confirming the applicability of SNOMED CT in general and its post-coordinated expressions in particular as a viable intermediate mapping step. Resulting in an agreement of 84,00 % between the newly developed approach and the manual mapping, it becomes obvious that the present approach has the potential to be used in everyday medical practice.

Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas.

Background: Patients with non-small cell lung cancer (NSCLC) harboring a ROS proto-oncogene 1 (ROS1)-rearrangement respond to treatment with ROS1 inhibitors. To distinguish these rare cases, screening with immunohistochemistry (IHC) for ROS1 protein expression has been suggested. However, the reliability of such an assay and the comparability of the antibody clones has been debated. Therefore we evaluated the diagnostic performance of current detection strategies for ROS1-rearrangement in two NSCLC-patient cohorts. Methods: Resected tissue samples, retrospectively collected from consecutive NSCLC-patients surgically treated at Uppsala University Hospital were incorporated into tissue microarrays [all n=676, adenocarcinomas (AC) n=401, squamous cell carcinomas (SCC) n=213, other NSCLC n=62]. ROS1-rearrangements were detected using fluorescence in situ hybridization (FISH) (Abbott Molecular; ZytoVision). In parallel, ROS1 protein expression was detected using IHC with three antibody clones (D4D6, SP384, EPMGHR2) and accuracy, sensitivity, and specificity were determined. Gene expression microarray data (Affymetrix) and RNA-sequencing data were available for a subset of patients. NanoString analyses were performed for samples with positive or ambiguous results (n=21). Results: Using FISH, 2/630 (0.3% all NSCLC; 0.5% non-squamous NSCLC) cases were positive for ROS1 fusion. Additionally, nine cases demonstrated ambiguous FISH results. Using IHC, ROS1 protein expression was detected in 24/665 (3.6% all NSCLC; 5.1% non-squamous NSCLC) cases with clone D4D6, in 18/639 (2.8% all NSCLC; 3.9% non-squamous NSCLC) cases with clone SP384, and in 1/593 (0.2% all NSCLC; 0.3% non-squamous NSCLC) case with clone EPMGHR2. Elevated RNA-levels were seen in 19/369 (5.1%) cases (Affymetrix and RNA-sequencing combined). The overlap of positive results between the assays was poor. Only one of the FISH-positive cases was positive with all antibodies and demonstrated high RNA-expression. This rearrangement was confirmed in the NanoString-assay and also in the RNA-sequencing data. Other cases with high protein/RNA-expression or ambiguous FISH were negative in the NanoString-assay. Conclusions: The occurrence of ROS1 fusions is low in our cohorts. The IHC assays detected the fusions, but the accuracy varied depending on the clone. The presumably false-positive and uncertain FISH results questions this method for detection of ROS1-rearrangements. Thus, when IHC is used for screening, transcript-based assays are preferable for validation in clinical diagnostics.

Spatial Distribution of Immune Cells in Head and Neck Squamous Cell Carcinomas.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) have a very moderate response rate to immune checkpoint inhibitor (ICI) treatment compared to other cancer types. Lacking predictive markers for treatment response, we analyzed the immune status of HNSCC and assessed the spatial distribution of immune cells. MATERIALS AND METHODS: Via assessing hematoxylin-eosin (H&E) stains, we divided HNSCCs by the immune cell distribution in hot, cold, and excluded tumors. For each group, each with 10 tumors, we performed serial immunohistochemical (IHC) staining of the immune cell markers, checkpoint molecules, and immune regulators. RESULTS: The spatial distributions were different for each immune cell type, allocating regulatory T cells (Tregs) and CD11b cells predominantly in the stroma. CD4 and CD8 cells were present either in the tumor stroma or between cancer cells. Interestingly, the expressions of PD-1 (programmed cell death 1 receptor) and PD-L1 (programmed death-ligand 1) were higher in hot tumors in comparison to cold and excluded tumors. The expression of pSMAD [indicating active transforming growth factor beta (TGF-ß)] was higher in excluded tumors. CONCLUSION: Different immune cell distribution patterns within tumors might be crucial for ICI treatment response since hot tumors have the highest expressions of PD-1 and PD-L1. TGF-ß might be a key regulator for immune cell distribution and a promising therapeutic target that determines the formation of hot or excluded immune patterns.

Delta-Like Protein 3 Expression in Paired Chemonaive and Chemorelapsed Small Cell Lung Cancer Samples.

Rovalpituzumab tesirine (Rova-T), an antibody-drug conjugate directed against Delta-like protein 3 (DLL3), is under development for patients with small cell lung cancer (SCLC). DLL3 is expressed on the majority of SCLC samples. Because SCLC is rarely biopsied in the course of disease, data regarding DLL3 expression in relapses is not available. The aim of this study was to investigate the expression of DLL3 in chemorelapsed (but untreated with Rova-T) SCLC samples and compare the results with chemonaive counterparts. Two evaluation methods to assess DLL3 expression were explored. Additionally, we assessed if DLL3 expression of chemorelapsed and/or chemonaive samples has prognostic impact and if it correlates with other clinicopathological data. The study included 30 paired SCLC samples, which were stained with an anti DLL3 antibody. DLL3 expression was assessed using tumor proportion score (TPS) and H-score and was categorized as DLL3 low (TPS < 50%, H-score ≤ 150) and DLL3 high (TPS ≥ 50%, H-score > 150). Expression data were correlated with clinicopathological characteristics. Kaplan-Meier curves were used to illustrate overall survival (OS) depending on DLL3 expression in chemonaive and chemorelapsed samples, respectively, and depending on dynamics of expression during course of therapy. DLL3 was expressed in 86.6% chemonaive and 80% chemorelapsed SCLC samples without significant differences between the two groups. However, the extent of expression varied in a substantial proportion of pairs (36.6% with TPS, 43.3% with H-score), defined as a shift from low to high or high to low expression. TPS and H-score provided comparable results. There were no profound correlations with clinicopathological data. Survival analysis revealed a trend toward a more favorable OS in DLL low-expressing chemonaive SCLC (p = 0.57) and, in turn, in DLL3 high-expressing chemorelapsed SCLC (p = 0.42) as well as in SCLC demonstrating a shift from low to high expression (p = 0.56) without being statistically significant. This is the first study to investigate DLL3 expression in a large cohort of rare paired chemonaive-chemorelapsed SCLC specimens. Comparative analysis revealed that DLL3 expression was not stable during the course of therapy, suggesting therapy-based alterations. Unlike in chemonaive samples, a high DLL3 expression in chemorelapsed samples indicated a trend for a more favorable prognosis. Our results highlight the importance to investigate DLL3 in latest chemorelapsed SCLC tumor tissue.

Cracking it - successful mRNA extraction for digital gene expression analysis from decalcified, formalin-fixed and paraffin-embedded bone tissue.

With the advance of precision medicine, the availability of tumor tissue for molecular analysis has become a limiting factor. This is particularly the case for bone metastases which are frequently occurring in cancer types such as prostate cancer. Due to the necessary decalcification process it was long thought that transcriptome analysis will not be feasible from decalcified formalin-fixed, paraffin-embedded (DFFPE) in a large manner. Here we demonstrate that mRNA extraction from DFFPE is feasible, quick, robust and reproducible and that decalcification does not hamper subsequent gene expression analysis. This might assist in implementing transcriptome analysis from DFFPE into every day practice.

Performance of Different Diagnostic PD-L1 Clones in Head and Neck Squamous Cell Carcinoma.

Background: The approval of immune checkpoint inhibitors in combination with specific diagnostic biomarkers presents new challenges to pathologists as tumor tissue needs to be tested for expression of programmed death-ligand 1 (PD-L1) for a variety of indications. As there is currently no requirement to use companion diagnostic assays for PD-L1 testing in Germany different clones are used in daily routine. While the correlation of staining results has been tested in various entities, there is no data for head and neck squamous cell carcinomas (HNSCC) so far. Methods: We tested five different PD-L1 clones (SP263, SP142, E1L3N, 22-8, 22C3) on primary HNSCC tumor tissue of 75 patients in the form of tissue microarrays. Stainings of both immune and tumor cells were then assessed and quantified by pathologists to simulate real-world routine diagnostics. The results were analyzed descriptively and the resulting staining pattern across patients was further investigated by principal component analysis and non-negative matrix factorization clustering. Results: Percentages of positive immune and tumor cells varied greatly. Both the resulting combined positive score as well as the eligibility for certain checkpoint inhibitor regimens was therefore strongly dependent on the choice of the antibody. No relevant co-clustering and low similarity of relative staining patterns across patients was found for the different antibodies. Conclusions: Performance of different diagnostic anti PD-L1 antibody clones in HNSCC is less robust and interchangeable compared to reported data from other tumor entities. Determination of PD-L1 expression is critical for therapeutic decision making and may be aided by back-to-back testing of different PD-L1 clones.

Immunologic "Cold" Squamous Cell Carcinomas of the Head and Neck Are Associated With an Unfavorable Prognosis.

Background: Head and neck squamous cell carcinoma (HNSCC) represents a common cancer worldwide. Past therapeutic advances have not significantly improved HNSCC prognosis. Therefore, it is necessary to further stratify HNSCC, especially with recent advances in tumor immunology. Methods: Tissue microarrays were assembled from tumor tissue samples and were complemented with comprehensive clinicopathological data of n = 419 patients. H&E whole slides from resection specimen (n = 289) were categorized according to their immune cell infiltrate as "hot," "cold," or "excluded." Results: Investigating tumor immune cell patterns, we found significant differences in survival rates. Immunologic "hot" and "excluded" HNSCCs are associated with better overall survival than "cold" HNSCC patients (p < 0.05). Interestingly, the percentage of all three patterns is nearly identical in p16 positive and negative HNSCCs. Conclusions: Using a plain histological H&E approach to categorize HNSCC as being immunologic "hot," "cold," or "excluded" can offer a forecast of patients' prognosis and may thus aid as a potential prognostic tool in routine pathology reports. This "hot-cold-excluded" scheme needs to be applied to more HNSCC cohorts and possibly to other cancer types to determine prognostic meaning, e.g., regarding OS or DFS. Furthermore, our cohort reflects epidemiological data in the national, European, and international context. It may, therefore, be of use for future HNSCC characterization.

PD-L1 amplification is associated with an immune cell rich phenotype in squamous cell cancer of the lung.

Gene amplification is considered to be one responsible cause for upregulation of Programmed Death Ligand-1 (PD-L1) in non-small cell lung cancer (NSCLC) and to represent a specific molecular subgroup possibly associated with immunotherapy response. Our aim was to analyze the frequency of PD-L1 amplification, its relation to PD-L1 mRNA and protein expression, and to characterize the immune microenvironment of amplified cases. The study was based on two independent NSCLC cohorts, including 354 and 349 cases, respectively. Tissue microarrays were used to evaluate PD-L1 amplification by FISH and PD-L1 protein by immunohistochemistry. Immune infiltrates were characterized immunohistochemically by a panel of immune markers (CD3, CD4, CD8, PD-1, Foxp3, CD20, CD138, CD168, CD45RO, NKp46). Mutational status was determined by targeted sequencing. RNAseq data was available for 197 patients. PD-L1 amplification was detected in 4.5% of all evaluable cases. PD-L1 amplification correlated only weakly with mRNA and protein expression. About  37% of amplified cases were negative for PD-L1 protein. PD-L1 amplification did not show any association with the mutational status. In squamous cell cancer, PD-L1 amplified cases were enriched among patients with high tumoral immune cell infiltration and showed gene expression profiles related to immune exhaustion. In conclusion, PD-L1 amplification correlates with PD-L1 expression in squamous cell cancer and was associated with an immune cell rich tumor phenotype. The correlative findings help to understand the role of PD-L1 amplification as an important immune escape mechanism in NSCLC and suggest the need to further evaluate PD-L1 amplification as predictive biomarker for checkpoint inhibitor therapy.

[Mycobacterium szulgai as positive control helps to detect contaminations in the detection of Mycobacterium tuberculosis-complex in formalin fixed, paraffin embedded tissues by 16SrDNA PCR]. / Mycobacterium szulgai als Posititivkontrolle zur Detektion von Kontaminationen beim Nachweis des Mycobacterium tuberculosis-Komplexes durch eine spezifische 16S-rDNA-PCR an FFPE-Material.

The detection of Mycobacterium tuberculosis complex DNA by PCR using formalin-fixed paraffin-embedded material has become an integral part of molecular-pathological diagnostics. We describe an approach that enables the detection of contamination by using Mycobacterium szulgai as a positive control, contributing to the reduction of false-positive results.

[Cryptogenic organizing pneumonia versus secondary organizing pneumonia]. / Kryptogen organisierende Pneumonie versus sekundäre organisierende Pneumonie.

Organizing pneumonia (OP) describes a histological pattern of acute or subacute lung damage. Clinically, patients present with cough, fever, and dyspnea. A distinction is made between idiopathic or cryptogenic organizing pneumonia (COP) and secondary organizing pneumonia (OP). In COP, neither clinical/radiological nor histological causes can be determined. It is classified as an interstitial idiopathic pneumonia (IIP) according to the criteria of the American Thoracic Society (ATS) and the European Respiratory Society (ERS). Secondary organizing pneumonia has a known triggering mechanism, such as infectious agents, certain medications, or concomitant symptoms of other primary pulmonary diseases and diseases of other organ systems. Common to both forms is the histological picture of intra-alveolar mesenchymal buds. These are myofibroblast proliferates that branch out along the alveolar spaces. They are usually accompanied by a moderate interstitial and alveolar, chronic, and macrophage-rich inflammatory cell infiltrate. The most important differential diagnosis is common interstitial pneumonia (UIP). It also shows fibroblast proliferates, which are, however, located in the interstitium. The correct classification of an IIP as a COP by means of clinical, radiological, and histological findings is essential, since the COP, in contrast to the UIP, responds very well to corticosteroids and therefore has an excellent prognosis compared to the UIP. The course of secondary organizing pneumonia depends on the respective underlying disease. Here it is important for the pathologist to correctly identify potential accompanying histological characteristics in order to be able to provide clues to a possible cause of OP.

Impact of Spatially Heterogeneous Trop-2 Expression on Prognosis in Oral Squamous Cell Carcinoma.

Oral cancer often presents with aggressive behavior and a high risk of recurrence and metastasis. For oral squamous cell carcinoma (OSCC), which is the most frequent histological subtype, therapy strategies include surgery, radiation therapy, chemotherapy, immune checkpoint inhibitors, and EGFR inhibitors. Recently, a Trop-2 antibody-drug conjugate (ADC) has been approved in the United States of America for the treatment of advanced triple-negative breast cancer. However, this ADC has also been tested in other solid tumors including head & neck squamous cell carcinoma. The prognostic impact of Trop-2 has already been reported for several cancers. We studied the prognostic influence of Trop-2 protein expression on OSCC patients' survival. The cohort comprised n = 229 OSCC patients with available archived tumor tissue and corresponding non-neoplastic oral mucosa tissue. Using immunohistochemistry, we investigated Trop-2 expression in both the central and peripheral regions of each tumor and in corresponding non-neoplastic oral mucosa. In patients suffering from OSCC with combined high central and low peripheral Trop-2 expression, five-year overall survival (OS) was 41.2%, whereas 55.6% of OSCC patients who presented lower central and/or higher peripheral tumoral Trop-2 expression were alive after five years (p = 0.075). In multivariate Cox regression, the expression pattern of high central tumoral and lower peripheral Trop-2 expression was significantly correlated with impaired OS (HR = 1.802, 95%-CI: 1.134-2.864; p = 0.013) and recurrence-free survival (RFS) (HR = 1.633, 95%-CI: 1.042-2.560; p = 0.033), respectively, when adjusting for co-variables. Hence, Trop-2 may serve as an independent prognostic biomarker in OSCC. In subsequent studies, the pathophysiological meaning of downregulated Trop-2 expression in the OSCC periphery has to be analyzed.

The predictive power of CD3+ T cell infiltration of oral squamous cell tumors is limited to non-diabetic patients.

Diabetes mellitus type II (DM) and immune cell infiltration determine patient outcome in many tumor entities. Here we studied a possible link between the metabolic and immune cell status of OSCC patients. Glucose transporter (GLUT) 1 mRNA expression was elevated in all tumor samples, whereas other glycolytic markers such as lactate dehydrogenase (LDH) A or monocarboxylate transporter (MCT) 1 were increased in tumor samples from patients with diabetes and these patients had a significantly worse prognosis compared to non-diabetic patients. Analyses of immune cell infiltration in tumors from diabetic and non-diabetic patients revealed an increased leukocyte (CD45+) infiltration compared to normal mucosa only in non-diabetic patients. In line, the amount of CD3+ T cells per mm2 tumor tissue, was elevated in patients without diabetes and crucial for patient outcome in OSCC patients without diabetes, as compared to healthy mucosa using fluorescence immunohistochemistry in tissue microarrays of 229 patients. Our results demonstrate that diabetes is a prognostic factor for OSCC patients and associates with decreased leukocyte and CD3+ infiltration indicating that metabolic differences between diabetic and non-diabetic patients may alter tumor-infiltrating T cells and thereby determine patient outcome.

Recurrent HNSCC Harbor an Immunosuppressive Tumor Immune Microenvironment Suggesting Successful Tumor Immune Evasion.

PURPOSE: Recurrent tumors (RT) of head and neck squamous cell carcinoma (HNSCC) occur in up to 60%, with poor therapeutic response and detrimental prognosis. We hypothesized that HNSCC RTs successfully evade antitumor immune response and aimed to reveal tumor immune microenvironment (TIME) changes of primary tumors (PT) and corresponding RTs. EXPERIMENTAL DESIGN: Tumor-infiltrating leukocytes (TIL) of 300 PTs and 108 RTs from two large independent and clinically well-characterized HNSCC cohorts [discovery cohort (DC), validation cohort (VD)] were compared by IHC. mRNA expression analysis of 730 immune-related genes was performed for 18 PTs and RTs after adjuvant chemoradiotherapy (CRT). The effect of chemotherapy and radiation resistance was assessed with an in vitro spheroid/immunocyte coculture model. RESULTS: TIME analysis revealed overall decrease of TILs with significant loss of CD8+ T cells (DC P = 0.045/VC P < 0.0001) and B lymphocytes (DC P = 0.036/VC P < 0.0001) in RTs compared with PTs in both cohorts. Decrease predominantly occurred in RTs after CRT. Gene expression analysis confirmed loss of TILs (P = 0.0004) and B lymphocytes (P < 0.0001) and showed relative increase of neutrophils (P = 0.018), macrophages (P < 0.0001), dendritic cells (P = 0.0002), and mast cells (P = 0.0057) as well as lower overall expression of immune-related genes (P = 0.018) in RTs after CRT. Genes involved in B-lymphocyte functions and number of tertiary lymphoid structures showed the strongest decrease. SPP1 and MAPK1 were upregulated in vivo and in vitro, indicating their potential suitability as therapeutic targets in CRT resistance. CONCLUSIONS: HNSCC RTs have an immunosuppressive TIME, which is particularly apparent after adjuvant CRT and might substantially contribute to poor therapeutic response and prognosis.

In silico analysis reveals EP300 as a panCancer inhibitor of anti-tumor immune response via metabolic modulation.

The tumor immune microenvironment (TIME) of head and neck squamous cell carcinomas (HNSCC) and other solid malignancies is a key determinant of therapy response and prognosis. Among other factors, it is shaped by the tumor mutational burden and defects in DNA repair enzymes. Based on the TCGA database we aimed to define specific, altered genes associated with different TIME types, which might represent new predictive markers or targets for immuno-therapeutic approaches. The HNSCC cohort of the TCGA database was used to define 3 TIME types (immune-activated, immune-suppressed, immune-absent) according to expression of immune-related genes. Mutation frequencies were correlated to the 3 TIME types. Overall survival was best in the immune-activated group. 9 genes were significantly differentially mutated in the 3 TIME types with strongest differences for TP53 and the histone-acetyltransferase EP300. Mutations in EP300 correlated with an immune-activated TIME. In panCancer analyses anti-tumor immune activity was increased in EP300 mutated esophageal, stomach and prostate cancers. Downregulation of EP300 gene expression was associated with higher anti-tumor immunity in most solid malignancies. Since EP300 is a promoter of glycolysis, which negatively affects anti-tumor immune response, we analyzed the association of EP300 with tumor metabolism. PanCancer tumor metabolism was strongly shifted towards oxidative phosphorylation in EP300 downregulated tumors. In silico analyses of of publicly available in vitro data showed a decrease of glycolysis-associated genes after treatment with the EP300 inhibitor C646. Our study reveals associations of specific gene alterations with different TIME types. In detail, we defined EP300 as a panCancer inhibitor of the TIME most likely via metabolic modulation. In this context EP300 represents a promising predictive biomarker and an immuno-therapeutic target.

EVI1 as a Marker for Lymph Node Metastasis in HNSCC.

BACKGROUND: HNSCC is the sixth most common cancer in humans and has still a very poor prognosis. The treatment methods so far are very often associated with mutilation and impairment in the quality of life. Except for p16 expression, there are no reliable prognostic markers in HNSCC so far. Ecotropic Viral Integration Site 1 (EVI1) is a well-described prognostic marker in leukemia and different types of solid cancers. In these, a high EVI1 expression is associated with a poor prognosis. In HNSCC, it is not known so far if EVI1 has any prognostic relevance. MATERIALS AND METHODS: We used our representative tissue cohort of 389 primary HNSCCs, of which 57.2% had one or more lymph node metastases. Here EVI1 expression was analyzed via immunohistochemistry and correlated with the clinical characteristics of these patients. RESULTS: Although in HNSCC EVI1 expression does not predict poor survival, a high EVI1 expression in the primary tumor correlates with a lymph node metastatic disease. CONCLUSION: Consequently, EVI1 may serve as a biomarker to predict an occult lymph node metastasis in a clinical nodal negative (cN0) HNSCC.

Increased mediator complex subunit CDK19 expression associates with aggressive prostate cancer.

The Mediator complex is a transcriptional regulator interacting with transcription factors and RNA-polymerase-II. Recently, we identified its subunit CDK19 to be specifically expressed in prostate cancer (PCa) and to be functionally implicated in PCa aggressiveness. Aim of our study was to comprehensively characterize the protein expression of CDK19 and its paralog CDK8 in PCa. We performed immunohistochemistry (IHC) for CDK19/CDK8 on a large cohort including needle biopsies from 202 patients, 799 primary tumor foci of radical prostatectomy specimens from 415 patients, 120 locally advanced tumor foci obtained by palliative transurethral resection, 140 lymph node metastases, 67 distant metastases and 82 benigns. Primary tumors were stained for the proliferation marker Ki67, androgen receptor (AR) and ERG. For 376 patients, clinic-pathologic data were available. Primary endpoint was disease-recurrence-free survival (DFS). Nuclear CDK19 and CDK8 expression increases during progression showing the highest intensity in metastatic and castration-resistant tumors. High CDK19 expression on primary tumors correlates with DFS independently from Gleason grade and PSA. Five-year-DFS rates of patients with primary tumors expressing no, moderate and high CDK19 are 73.7, 56.9 and 30.4%, respectively. CDK19 correlates with Gleason grade, T-stage, Ki67 proliferation-index, nuclear AR expression and ERG-status. Therapeutic options for metastatic and castration-resistant PCa remain limited. In the current study, we confirmed an important role of the Mediator subunit CDK19 in advanced PCa supporting current developments to target CDK19 and its paralog CDK8. Furthermore, CDK19 protein expression has the potential to predict disease recurrence independently from established biomarkers thus contributing to individual management for PCa patients.

Complex Formation with Monomeric α-Tubulin and Importin 13 Fosters c-Jun Protein Stability and Is Required for c-Jun's Nuclear Translocation and Activity.

Microtubules are highly dynamic structures, which consist of α- and ß-tubulin heterodimers. They are essential for a number of cellular processes, including intracellular trafficking and mitosis. Tubulin-binding chemotherapeutics are used to treat different types of tumors, including malignant melanoma. The transcription factor c-Jun is a central driver of melanoma development and progression. Here, we identify the microtubule network as a main regulator of c-Jun activity. Monomeric α-tubulin fosters c-Jun protein stability by protein-protein interaction. In addition, this complex formation is necessary for c-Jun's nuclear localization sequence binding to importin 13, and consequent nuclear import and activity of c-Jun. A reduction in monomeric α-tubulin levels by treatment with the chemotherapeutic paclitaxel resulted in a decline in the nuclear accumulation of c-Jun in melanoma cells in an experimental murine model and in patients' tissues. These findings add important knowledge to the mechanism of the action of microtubule-targeting drugs and indicate the newly discovered regulation of c-Jun by the microtubule cytoskeleton as a novel therapeutic target for melanoma and potentially also other types of cancer.

Tumor microenvironment modulation enhances immunologic benefit of chemoradiotherapy.

BACKGROUND: Chemoradiotherapy (CRT) remains one of the most common cancer treatment modalities, and recent data suggest that CRT is maximally effective when there is generation of an anti-tumoral immune response. However, CRT has also been shown to promote immunosuppressive mechanisms which must be blocked or reversed to maximize its immune stimulating effects. METHODS: Therefore, using a preclinical model of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), we developed a clinically relevant therapy combining CRT and two existing immunomodulatory drugs: cyclophosphamide (CTX) and the small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). In this model, we treated the syngeneic HPV-HNSCC mEER tumor-bearing mice with fractionated (10 fractions of 3 Gy) tumor-directed radiation and weekly cisplatin administration. We compared the immune responses induced by CRT and those induced by combinatory treatment (CRT + CTX/L-NIL) with flow cytometry, quantitative multiplex immunofluorescence and by profiling immune-related gene expression changes. RESULTS: We show that combination treatment favorably remodels the tumor myeloid immune microenvironment including an increase in anti-tumor immune cell types (inflammatory monocytes and M1-like macrophages) and a decrease in immunosuppressive granulocytic myeloid-derived suppressor cells (MDSCs). Intratumoral T cell infiltration and tumor antigen specificity of T cells were also improved, including a 31.8-fold increase in the CD8+ T cell/ regulatory T cell ratio and a significant increase in tumor antigen-specific CD8+ T cells compared to CRT alone. CTX/LNIL immunomodulation was also shown to significantly improve CRT efficacy, leading to rejection of 21% established tumors in a CD8-dependent manner. CONCLUSIONS: Overall, these data show that modulation of the tumor immune microenvironment with CTX/L-NIL enhances susceptibility of treatment-refractory tumors to CRT. The combination of tumor immune microenvironment modulation with CRT constitutes a translationally relevant approach to enhance CRT efficacy through enhanced immune activation.