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Androgen deprivation therapy has a central role in the treatment of advanced prostate cancer, often causing initial tumour remission before increasing independence from signal transduction mechanisms of the androgen receptor and then eventual disease progression. Novel treatment approaches are urgently needed, but only a fraction of promising drug candidates from the laboratory will eventually reach clinical approval, highlighting the demand for critical assessment of current preclinical models. Such models include standard, genetically modified and patient-derived cell lines, spheroid and organoid culture models, scaffold and hydrogel cultures, tissue slices, tumour xenograft models, patient-derived xenograft and circulating tumour cell eXplant models as well as transgenic and knockout mouse models. These models need to account for inter-patient and intra-patient heterogeneity, the acquisition of primary or secondary resistance, the interaction of tumour cells with their microenvironment, which make crucial contributions to tumour progression and resistance, as well as the effects of the 3D tissue network on drug penetration, bioavailability and efficacy.

Progression of prostate cancer (PCa) is characterized by metastasis and castration resistance after response to androgen deprivation. Therapeutic options are limited, causing high morbidity and lethality. Recent work reported pro-oncogenic implications of the Mediator subunits cyclin-dependent kinase (CDK) 8 and 19 for the progression of PCa. The current study explored the underlying molecular mechanisms of CDK8/CDK19 and tested effects of novel CDK8/CDK19 inhibitors. PC3, DU145, LNCaP, and androgen-independent LNCaP Abl were used for in vitro experiments. Two inhibitors and CDK19 overexpression were used to modify CDK8/CDK19 activity. MTT assay, propidium iodide staining, wound healing assay, Boyden chamber assay, and adhesion assay were used to investigate cell viability, cell cycle, migration, and adhesion, respectively. Peptide-kinase screen using the PamGene platform was conducted to identify phosphorylated targets. Combining CDK8/CDK19 inhibitors with anti-androgens led to synergistic antiproliferative effects and sensitized androgen-independent cells to bicalutamide. CDK8/CDK19 inhibition resulted in reduced migration and increased collagen I-dependent adhesion. Phosphorylation of multiple peptides linked to cancer progression was identified to be dependent on CDK8/CDK19. In summary, this study substantially supports recent findings on CDK8/CDK19 in PCa progression. These findings contribute to a better understanding of underlying pro-oncogenic effects, which is needed to develop CDK8/CDK19 as a therapeutic target in PCa.

Purpose:The Mediator complex is a multiprotein assembly, which serves as a hub for diverse signaling pathways to regulate gene expression. Because gene expression is frequently altered in cancer, a systematic understanding of the Mediator complex in malignancies could foster the development of novel targeted therapeutic approaches.Experimental Design:We performed a systematic deconvolution of the Mediator subunit expression profiles across 23 cancer entities (n = 8,568) using data from The Cancer Genome Atlas (TCGA). Prostate cancer–specific findings were validated in two publicly available gene expression cohorts and a large cohort of primary and advanced prostate cancer (n = 622) stained by immunohistochemistry. The role of CDK19 and CDK8 was evaluated by siRNA-mediated gene knockdown and inhibitor treatment in prostate cancer cell lines with functional assays and gene expression analysis by RNAseq.Results:Cluster analysis of TCGA expression data segregated tumor entities, indicating tumor-type–specific Mediator complex compositions. Only prostate cancer was marked by high expression of CDK19. In primary prostate cancer, CDK19 was associated with increased aggressiveness and shorter disease-free survival. During cancer progression, highest levels of CDK19 and of its paralog CDK8 were present in metastases. In vitro, inhibition of CDK19 and CDK8 by knockdown or treatment with a selective CDK8/CDK19 inhibitor significantly decreased migration and invasion.Conclusions:Our analysis revealed distinct transcriptional expression profiles of the Mediator complex across cancer entities indicating differential modes of transcriptional regulation. Moreover, it identified CDK19 and CDK8 to be specifically overexpressed during prostate cancer progression, highlighting their potential as novel therapeutic targets in advanced prostate cancer. Clin Cancer Res; 23(7); 1829–40. ©2016 AACR.

Supplementary Data from Pan-Cancer Analysis of the Mediator Complex Transcriptome Identifies CDK19 and CDK8 as Therapeutic Targets in Advanced Prostate Cancer

Supplementary Tables 1-4 from Lysine-Specific Demethylase 1 Is Strongly Expressed in Poorly Differentiated Neuroblastoma: Implications for Therapy

Aberrant epigenetic changes in DNA methylation and histone acetylation are hallmarks of most cancers, whereas histone methylation was previously considered to be irreversible and less versatile. Recently, several histone demethylases were identified catalyzing the removal of methyl groups from histone H3 lysine residues and thereby influencing gene expression. Neuroblastomas continue to remain a clinical challenge despite advances in multimodal therapy. Here, we address the functional significance of the chromatin-modifying enzyme lysine-specific demethylase 1 (LSD1) in neuroblastoma. LSD1 expression correlated with adverse outcome and was inversely correlated with differentiation in neuroblastic tumors. Differentiation of neuroblastoma cells resulted in down-regulation of LSD1. Small interfering RNA–mediated knockdown of LSD1 decreased cellular growth, induced expression of differentiation-associated genes, and increased target gene–specific H3K4 methylation. Moreover, LSD1 inhibition using monoamine oxidase inhibitors resulted in an increase of global H3K4 methylation and growth inhibition of neuroblastoma cells in vitro. Finally, targeting LSD1 reduced neuroblastoma xenograft growth in vivo. Here, we provide the first evidence that a histone demethylase, LSD1, is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells. We show that inhibition of LSD1 reprograms the transcriptome of neuroblastoma cells and inhibits neuroblastoma xenograft growth. Our results suggest that targeting histone demethylases may provide a novel option for cancer therapy. [Cancer Res 2009;69(5):2065–71]

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Supplementary Figure and Table Legends from Lysine-Specific Demethylase 1 Is Strongly Expressed in Poorly Differentiated Neuroblastoma: Implications for Therapy

PDF file - 57K, Significant decrease of p-(Tyr416)-SRC levels upon siRNA-mediated knockdown of IGF-1R in CME-1 synovial sarcoma cells.

PDF file - 70K, As an indirect proof of specific SRC-related action of dasatinib in synovial sarcoma cells, CME-1 did not display any significant effects upon dasatinib treatement after siRNA-mediated SRC-knockdown.

Supplementary Figures 1-4 from Lysine-Specific Demethylase 1 Is Strongly Expressed in Poorly Differentiated Neuroblastoma: Implications for Therapy

PDF file - 57K, Densitometric analysis of the top five phosphorylated kinases as found in phospho kinase arrays in 1273/99 and HS-SY-II synovial sarcoma cells. Flow cytometric analysis of three synovial sarcoma cell lines treated with different doses of dasatinib for 48 hours.

Colorectal cancer is a notorious disease, with almost half of the patients succumbing to the disease. The prevalence and incidence rates of colorectal cancer are increasing in many parts of the world, highlighting the need to discover new biomarkers for diagnosis and therapy. Caldesmon (CaD), an actin-binding protein that plays a significant role in controlling cell motility, has emerged as a promising biomarker. The CALD1 gene encodes CaD as multiple transcripts that mainly encode two protein isoforms: High-molecular-weight (h-CaD), expressed in smooth muscle, and low-molecular-weight (l-CaD), expressed in nonsmooth muscle cells. Most studies have suggested an oncogenic role of CaD in colorectal cancer, but the exact subcellular localization of the two CaD isoforms in tumor cells and stroma have not been clarified yet. Here, we analyzed tissue samples from 262 colorectal cancer patients by immunohistochemistry analysis using specific antibodies for l-CaD and h-CaD. The results showed elevated cytoplasmic expression levels of l-Cad in 187/262 (71.4%) cases. l-Cad was expressed at low levels in the normal colon mucosa and was also consistently expressed in the cancer-associated stroma of all cases, suggesting that it could play a role in modulating the tumor microenvironment. l-CaD expression in cancer cells was associated with preinvasive stages of cancer. Survival analysis indicated that patients with high l-CaD expression in tumor cells could respond poorly to selective chemotherapeutic 5FU, but not combination chemotherapy. h-CaD was expressed in colonic and vascular smooth muscle cells as expected and to a lesser extent in the tumor-associated stroma, but it was not expressed in the cancer cells or normal colon mucosal epithelial cells. Collectively, these data clarify how the expression patterns of CaD isoforms in colorectal cancer can have applications in the management of colorectal cancer patients.

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 only potentially curative treatment for lung adenocarcinoma patients remains complete resection of early-stage tumors. However, many patients develop recurrence and die of their disease despite curative surgery. Underlying mechanisms leading to establishment of systemic disease after complete resection are mostly unknown. We therefore aimed at identifying molecular signatures of resected lung adenocarcinomas associated with the risk of an early relapse. The study comprised 89 patients with totally resected stage IA–IIIA lung adenocarcinomas. Patients suffering from an early relapse within two years after surgery were compared to patients without a relapse in two years. Patients were clinically and molecular pathologically characterized. Tumor tissues were immunohistochemically analyzed for the expression of Ki67, CD45, CD4, CD8, PD1, PD-L1, PD-L2 and CD34, by Nanostring nCounter PanCancer Immune Profiling Panel as well as a comprehensive methylome profiling using the Infinium MethylationEPIC BeadChip. We detected differential DNA methylation patterns as well as significantly differentially expressed genes associated with an early relapse after complete resection. Especially, CD1A was identified as a potential biomarker, whose reduced expression is associated with an early relapse. These findings might help to develop biomarkers improving risk assessment and patient selection for adjuvant therapy as well as establish novel targeted therapeutic strategies.

Prostate cancer (PCa) poses a major public health problem in men. Metastatic PCa is incurable, and ultimately threatens the life of many patients. Mutations in tumor suppressor genes and oncogenes are important for PCa progression, whereas the role of epigenetic factors in prostate carcinogenesis is insufficiently examined. The histone demethylase KDM5C exerts important roles in tumorigenesis. KDM5C has been reported to be highly expressed in various cancer cell types, particularly in primary PCa. Here, we could show that KDM5C is highly upregulated in metastatic PCa. Functionally, in KDM5C knockdown cells migratory and invasion capacity was reduced. Interestingly, modulation of KDM5C expression influences several EMT signaling pathways (e.g., Akt/mTOR), expression of EMT transcription factors, epigenetic modifiers, and miR-205, resulting in increased expression of E-cadherin and reduced expression of N-cadherin. Mouse xenografts of KDM5C knockdown cells showed reduced tumor growth. In addition, the Akt/mTOR pathway is one of the classic signaling pathways to mediate tumor metabolic homeostasis, which is beneficial for tumor growth and metastasis. Taken together, our findings indicate that a combination of a selective KDM5C- and Akt/mTOR-inhibitor might be a new promising therapeutic strategy to reduce metastatic burden in PCa.

Colorectal cancer (CRC) is a devastating disease, mainly because of metastasis. As a result, there is a need to better understand the molecular basis of invasion and metastasis and to identify new biomarkers and therapeutic targets to aid in managing these tumors. The actin cytoskeleton and actin-binding proteins are known to play an important role in the process of cancer metastasis because they control and execute essential steps in cell motility and contractility as well as cell division. Caldesmon (CaD) is an actin-binding protein encoded by the CALD1 gene as multiple transcripts that mainly encode two protein isoforms: High-molecular-weight CaD, expressed in smooth muscle, and low-molecular weight CaD (l-CaD), expressed in nonsmooth muscle cells. According to our comprehensive review of the literature, CaD, particularly l-CaD, plays a key role in the development, metastasis, and resistance to chemoradiotherapy in colorectal, breast, and urinary bladder cancers and gliomas, among other malignancies. CaD is involved in many aspects of the carcinogenic hallmarks, including epithelial mesenchymal transition

Nucleoporins as components of the nuclear pore complex (NCP) are known for regulating nuclear-cytoplasmatic transport. Recently, the nucleoporin POM121 was found to have an important impact on intranuclear translocation of prostate cancer (PCa)-specific tumor drivers including the androgen receptor (AR). The aim of our study was to assess the potential of POM121 as a prognostic biomarker.Therefore, we performed immunohistochemistry (IHC) for POM121 on a large clinically, well characterized PCa tissue cohort comprising benign prostatic samples, radical prostatectomy (RPE) samples, lymph node metastases, local recurrent tumors and distant metastases of 289 patients. Using a semi automated tissue image analysis software we evaluated POM121 protein expression level based on IHC.We could show that POM121 expression increases during tumor progression. Expression levels were significantly higher in primary tumors compared to benign samples (P = 0.001), and substantially higher in advanced tumors (P0.001) and in distant metastases (P = 0.006) compared to primary tumors. Furthermore, POM121 expression predicts biochemical recurrence free survival (BFS) after surgery independent of the WHO group and other clinicopathological markers. 5-years BFS with primary tumors lacking POM121 and expressing POM121 was 88.8% and 68.9%, respectively.Our study reveals the potential of POM121 as a potential biomarker for PCa, predicting BFS independent of other common clinicopathological parameters. Furthermore, POM121 might be a new targetable structure for patients suffering from advanced PCa.

Cyclin-dependent kinase (CDK) 7-mediated phosphorylation of Mediator-complex subunit 1 (MED1) enhances androgen receptor (AR) activity in prostate cancer (PCa). Hyperactive AR-signalling plays a key role for the development of castration resistance. Several CDK7 inhibitors are currently under investigation in Phase I/II trials addressing solid tumours, including PCa. Aim of this study was to characterize the CDK7/phospho-(p)MED1 axis in human tissue. Immunohistochemistry was performed on 595 PCa samples including 394 primary tumour foci obtained by radical prostatectomy (RP), 64 advanced or recurrent tumours obtained by palliative transurethral resection of the prostate (pTUR), 65 lymph node metastases (LNM), 35 distant metastases (DM) and 36 benign samples. CDK7 is expressed in 79.3% of PCa tissues and protein levels are significantly higher in LNM, pTUR and DM and lower in benign tissues compared to primary tumours. CDK7 and pMED1 expression show strong positive correlation. High expression of CDK7 associated with shorter 5-year biochemical recurrence-free-survival (63.0% vs. 85.0%) and reduced survival persists when adjusted for T-Stage, nodal status, resection boundaries, grade group and pre-operative prostate-specific antigen in multivariate Cox-regression (hazard ratio 4.30; 95% CI, 1.43 to 12,40, P = 0.007). High CDK7 and pMED1 levels correlate with nuclear AR expression. CDK7 positive tumours harbour higher Ki67 expression indices and show more frequently positive ERG (ETS-related gene)-status. In conclusion, CDK7 is frequently expressed in human PCa and predicts disease recurrence after RP. Therapeutical inhibition of CDK7 might be a promising approach in treatment of advanced PCa.

The spectrum of somatic genetic variation in colorectal adenomas caused by biallelic pathogenic germline variants in the MSH3 gene, was comprehensively analysed to characterise mutational signatures and identify potential driver genes and pathways of MSH3-related tumourigenesis. Three patients from two families with MSH3-associated polyposis were included. Whole exome sequencing of nine adenomas and matched normal tissue was performed. The amount of somatic variants in the MSH3-deficient adenomas and the pattern of single nucleotide variants (SNVs) was similar to sporadic adenomas, whereas the fraction of small insertions/deletions (indels) (21–42% of all small variants) was significantly higher. Interestingly, pathogenic somatic APC variants were found in all but one adenoma. The vast majority (12/13) of these were di-, tetra-, or penta-base pair (bp) deletions. The fraction of APC indels was significantly higher than that reported in patients with familial adenomatous polyposis (FAP) (p < 0.01) or in sporadic adenomas (p < 0.0001). In MSH3-deficient adenomas, the occurrence of APC indels in a repetitive sequence context was significantly higher than in FAP patients (p < 0.01). In addition, the MSH3-deficient adenomas harboured one to five (recurrent) somatic variants in 13 established or candidate driver genes for early colorectal carcinogenesis, including ACVR2A and ARID genes. Our data suggest that MSH3-related colorectal carcinogenesis seems to follow the classical APC-driven pathway. In line with the specific function of MSH3 in the mismatch repair (MMR) system, we identified a characteristic APC mutational pattern in MSH3-deficient adenomas, and confirmed further driver genes for colorectal tumourigenesis.

Background: Lung cancer is the most frequent cause of cancer-related deaths worldwide. The clinical development of immune checkpoint blockade has dramatically changed the treatment paradigm for patients with lung cancer. Yet, an improved understanding of PD-1/PD-L1 checkpoint blockade-responsive biology is warranted. Methods: We aimed to identify the landscape of immune cell infiltration in primary lung adenocarcinoma (LUAD) in the context of tumoral PD-L1 expression and the extent of immune infiltration (“hot” vs. “cold” phenotype). The study comprises LUAD cases (n = 138) with “hot” (≥150 lymphocytes/HPF) and “cold” (&lt, 150 lymphocytes/HPF) tumor immune phenotype and positive (&gt, 50%) and negative (&lt, 1%) tumor PD-L1 expression, respectively. Tumor samples were immunohistochemically analyzed for expression of PD-L1, CD4, and CD8, and further investigated by transcriptome analysis. Results: Gene set enrichment analysis defined complement, IL-JAK-STAT signaling, KRAS signaling, inflammatory response, TNF-alpha signaling, interferon-gamma response, interferon-alpha response, and allograft rejection as significantly upregulated pathways in the PD-L1-positive hot subgroup. Additionally, we demonstrated that STAT1 is upregulated in the PD-L1-positive hot subgroup and KIT in the PD-L1-negative hot subgroup. Conclusion: The presented study illustrates novel aspects of PD-L1 regulation, with potential biological relevance, as well as relevance for immunotherapy response stratification.

Round robin testing is an important instrument for quality assurance. Increasingly, this also applies to the results of molecular diagnostics in pathology, which directly influence therapy decisions in precision oncology. In metastatic colorectal carcinoma (mCRC), the focus has been on detecting KRAS and NRAS mutations, whose absence allows therapy with EGFR blocking antibodies. Recently, BRAF has been added as another predictive marker, since mCRC patients with BRAF V600E mutation benefit significantly from treatment with encorafenib (a BRAF inhibitor) in combination with cetuximab (anti-EGFR antibody) after systemic therapy. Due to the approval of this treatment in 2020, it is a pre-requisite that BRAF V600E mutation detection in diagnostic pathologies is reliably performed. Therefore, this round robin test with BRAF V600E testing either by immunohistochemistry or molecular methods was performed. The round robin test results demonstrate that molecular BRAF V600E detection is currently clearly superior to immunohistochemical detection.Ringversuche sind ein wichtiges Instrument zur Qualitätssicherung. Dies betrifft in zunehmendem Maße auch die molekulare Diagnostik in der Pathologie, von deren Ergebnissen Therapieentscheidungen in der Präzisionsonkologie direkt abhängen. Beim metastasierten kolorektalen Karzinom (mKRK) stand bisher der Nachweis von KRAS-und NRAS-Mutationen im Vordergrund, deren Abwesenheit eine Therapie mit EGFR-blockierenden Antikörpern ermöglicht. Nun ist BRAF als weiterer prädiktiver Marker hinzugekommen, da mKRK Patienten mit einer BRAF-V600E-Mutation nach systemischer Vortherapie von einer Behandlung mit Encorafenib (einem BRAF-Inhibitor) in Kombination mit Cetuximab (Anti-EGFR-Antikörper) profitieren. Aufgrund der 2020 erfolgten Zulassung für diese Behandlung ist es wichtig, dass der diagnostische Nachweis einer BRAF-V600E-Mutation zuverlässig in den Pathologien durchgeführt werden kann. Daher wurde dieser Ringversuch durchgeführt, bei dem der Nachweis der BRAF-V600E-Mutation entweder mittels Immunhistochemie oder molekularer Verfahren erfolgen konnte. Die Ergebnisse des Ringversuchs belegen eindeutig, dass derzeit die molekulare BRAF-V600E-Bestimmung dem immunhistologischen Nachweis überlegen ist.

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.

Tripartite motif (TRIM) family proteins are post-translational protein modifiers with E3-ubiquitin ligase activity, thereby involved in various biological processes. The molecular mechanisms driving prostate cancer (PCa) bone metastasis (BM) are incompletely understood, and targetable genetic alterations are lacking in the majority of cases. Therefore, we aimed to explore the expression and potential functional relevance of 71 TRIM members in bone metastatic PCa. We performed transcriptome analysis of all human TRIM family members and 770 cancer-related genes in 29 localized PCa and 30 PCa BM using Nanostring. KEGG, STRING and Ubibrowser were used for further bioinformatic gene correlation and pathway enrichment analyses. Compared to localized tumors, six TRIMs are under-expressed while nine TRIMs are over-expressed in BM. The differentially expressed TRIM proteins are linked to TNF-, TGFβ-, PI3K/AKT- and HIF-1-signaling, and to features such as proteoglycans, platelet activation, adhesion and ECM-interaction based on correlation to cancer-related genes. The identification of TRIM-specific E3-ligase-substrates revealed insight into functional connections to oncogenes, tumor suppressors and cancer-related pathways including androgen receptor- and TGFβ signaling, cell cycle regulation and splicing. In summary, this is the first study that comprehensively and systematically characterizes the expression of all TRIM members in PCa BM. Our results describe post-translational protein modification as an important regulatory mechanism of oncogenes, tumor suppressors, and pathway molecules in PCa progression. Therefore, this study may provide evidence for novel therapeutic targets, in particular for the treatment or prevention of BM.

ObjectivesFormalin-fixed paraffin-embedded (FFPE) tissue is the standard material for diagnostic pathology but poses relevant hurdles to accurate protein extraction due to cross-linking and chemical alterations. While numerous extraction protocols and chemicals have been described, systematic comparative analyses are limited. Various parameters were thus investigated in their qualitative and quantitative effects on protein extraction (PE) efficacy. Special emphasis was put on preservation of membrane proteins (MP) as key subgroup of functionally relevant proteins.MethodsUsing the example of urothelial carcinoma, FFPE tissue sections were subjected to various deparaffinization, protein extraction and antigen retrieval protocols and buffers as well as different extraction techniques. Performance was measured by protein concentration and western blot analysis of cellular compartment markers as well as liquid chromatography-coupled mass spectrometry (LC–MS).ResultsCommercially available extraction buffers showed reduced extraction of MPs and came at considerably increased costs. On-slide extraction did not improve PE whereas several other preanalytical steps could be simplified. Systematic variation of temperature and exposure duration demonstrated a quantitatively relevant corridor of optimal antigen retrieval.ConclusionsPreanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.

Summary High-grade prostatic intraepithelial neoplasia (HGPIN) is a facultative precursor lesion of prostate cancer (PCa). Multifocal HGPIN in needle biopsies in the absence of PCa indicates a higher risk of cancer detection in subsequent biopsies. Therefore, a reliable diagnosis of HGPIN is of high clinical relevance guiding the management of patients with cancer-negative biopsies. Detection of HGPIN is merely based on morphological features while biomarkers aiding in the diagnosis of HGPIN and its differentiation from benign glands and other glandular lesions are lacking yet. Here, we investigated the expression of cyclin-dependent kinase 19 (CDK19) by immunohistochemistry on prostate needle biopsies of 140 patients who were all diagnosed with PCa using whole-tissue sections and compared CDK19 levels between HGPIN, PCa, and adjacent benign glands. In addition, CDK19 was compared with AMACR expression in a subset of intraductal carcinomas (IDCs) on radical prostatectomy (RP) specimens. HGPIN was present in 65.7% of biopsies and in 88% associated to adjacent PCa. CDK19 overexpression defined as moderate to high CDK19 expression visible at low magnification was found in 82.6% of HGPIN. In contrast, 89.3% of benign glands were CDK19-negative or demonstrated only low CDK19 expression highlighting a high sensitivity and specificity to accurately detect HGPIN based on CDK19 expression levels. CDK19 was overexpressed in 59% of PCa but did not correlate significantly with the expression of intermingled HGPIN. On RP, CDK19 and AMACR showed no significant difference in the detection rate of IDC. In summary, assessment of CDK19 facilitates accurate and simplified diagnosis of HGPIN with high sensitivity and specificity and aides the differentiation to non-neoplastic glandular alterations. Considering the high clinical significance of diagnosis HGPIN that still has a limited reproducibility among pathologists, we suggest CDK19 as diagnostic biomarker for HGPIN.

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.

Round robin testing is an important instrument for quality assurance. Increasingly, this also applies to the results of molecular diagnostics in pathology, which directly influence therapy decisions in precision oncology. In metastatic colorectal carcinoma (mCRC), the focus has been on detecting KRAS and NRAS mutations, whose absence allows therapy with EGFR blocking antibodies. Recently, BRAF has been added as another predictive marker, since mCRC patients with BRAF V600E mutation benefit significantly from treatment with encorafenib (a BRAF inhibitor) in combination with cetuximab (anti-EGFR antibody) after systemic therapy. Due to the approval of this treatment in 2020, it is a pre-requisite that BRAF V600E mutation detection in diagnostic pathologies is reliably performed. Therefore, this round robin test with BRAF V600E testing either by immunohistochemistry or molecular methods was performed. The round robin test results demonstrate that molecular BRAF V600E detection is currently clearly superior to immunohistochemical detection., Ringversuche sind ein wichtiges Instrument zur Qualitätssicherung. Dies betrifft in zunehmendem Maße auch die molekulare Diagnostik in der Pathologie, von deren Ergebnissen Therapieentscheidungen in der Präzisionsonkologie direkt abhängen. Beim metastasierten kolorektalen Karzinom (mKRK) stand bisher der Nachweis von KRAS-und NRAS-Mutationen im Vordergrund, deren Abwesenheit eine Therapie mit EGFR-blockierenden Antikörpern ermöglicht. Nun ist BRAF als weiterer prädiktiver Marker hinzugekommen, da mKRK Patienten mit einer BRAF-V600E-Mutation nach systemischer Vortherapie von einer Behandlung mit Encorafenib (einem BRAF-Inhibitor) in Kombination mit Cetuximab (Anti-EGFR-Antikörper) profitieren. Aufgrund der 2020 erfolgten Zulassung für diese Behandlung ist es wichtig, dass der diagnostische Nachweis einer BRAF-V600E-Mutation zuverlässig in den Pathologien durchgeführt werden kann. Daher wurde dieser Ringversuch durchgeführt, bei dem der Nachweis der BRAF-V600E-Mutation entweder mittels Immunhistochemie oder molekularer Verfahren erfolgen konnte. Die Ergebnisse des Ringversuchs belegen eindeutig, dass derzeit die molekulare BRAF-V600E-Bestimmung dem immunhistologischen Nachweis überlegen ist.

Background: Head and neck squamous cell carcinomas (HNSCCs) are among the most common cancers in humans worldwide and have a rather poor prognosis. TRIM24 has various intracellular functions and was identified in other cancer entities as a poor prognostic factor for patients. Methods: The expression of TRIM24 was evaluated by using immunohistochemistry. We used a large and representative cohort of 341 HNSCC patients. Data derived from immunohistochemistry evaluation was correlated with clinicopathological data from HNSCC patients. Results: The TRIM24 expression in HNSCC primary tumors is negatively correlated with the p16 status of the tumor tissues. Primary tumors of patients who developed a local recurrence were significantly more often positive for TRIM24. Kaplan–Meier analyses and Cox regression showed that patients with TRIM24 expressing tumors have significantly worse overall survival and progression-free survival and that TRIM24 expression is independent of other established risk factors. Conclusions: TRIM24 might be a new prognostic biomarker for the survival prognosis and early detection of local recurrences in HNSCC patients. It could be used for risk stratification of HNSCC patients and to identify those patients who are more prone to develop a local recurrence and therefore could profit from more frequent follow-up examinations.

Head and neck squamous cell carcinomas (HNSCC) are among the most common cancers worldwide and are associated with a poor prognosis for patients. Among HNSCC, those originating in the hypopharynx have the worst prognosis. The histone demethylase LSD1 has been shown to promote cancer initiation, progression, and relapse through various mechanisms and is upregulated in many cancer tissues. LSD1 physically interacts with SNAIL and is required for SNAIL mediated transcriptional repression. Previous studies of the prognostic value of LSD1 in HNSCC have been limited in their analysis of sub-sites, and a correlation between LSD1 and SNAIL has not been shown in HNSCC patient samples. Here we used a large, representative, and clinically well-characterized cohort of 339 HNSCC patients to investigate the co-expression of LSD1 and SNAIL and their prognostic value in all HNSCC using immunohistochemical staining. Elevated LSD1 expression correlated with advanced tumor stage and poor progression-free survival (PFS) in HNSCC originating in the hypopharynx. Overexpression of the transcription factor SNAIL independently correlated with worse overall survival (OS) and PFS in HNSCC in general and prominently in tumors of the hypopharynx. Furthermore, increased LSD1 expression significantly correlated with elevated SNAIL expression in patient samples. Therefore, the presented data implicates LSD1 and SNAIL as independent prognostic biomarkers.

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.

Die molekulare Landschaft des primaren und des metastasierten Prostatakarzinoms ist in den letzten Jahren gut charakterisiert worden. Das folgende Kapitel beleuchtet die molekularen Zusammenhange und zeigt pradiktive und prognostische Biomarker auf.