Targeting the glutamine metabolism to suppress cell proliferation in mesenchymal docetaxel-resistant prostate cancer.

Docetaxel (DX) serves as a palliative treatment option for metastatic prostate cancer (PCa). Despite initial remission, acquired DX resistance is inevitable. The mechanisms behind DX resistance have not yet been deciphered, but a mesenchymal phenotype is associated with DX resistance. Mesenchymal phenotypes have been linked to metabolic rewiring, obtaining most ATP production by oxidative phosphorylation (OXPHOS) powered substantially by glutamine (Gln). Likewise, Gln is known to play an essential role in modulating bioenergetic, redox homeostasis and autophagy. Herein, investigations of Gln deprivation on DX-sensitive and -resistant (DR) PCa cells revealed that the DR cell sub-lines were susceptible to Gln deprivation. Mechanistically, Gln deprivation reduced OXPHOS and ATP levels, causing a disturbance in cell cycle progression. Genetic and chemical inhibition of the Gln-metabolism key protein GLS1 could validate the Gln deprivation results, thereby representing a valid therapeutic target. Moreover, immunohistological investigation of GLS1 revealed a high-expressing GLS1 subgroup post-docetaxel failure, exhibiting low overall survival. This subgroup presents an intriguing opportunity for targeted therapy focusing on glutamine metabolism. Thus, these findings highlight a possible clinical rationale for the chemical inhibition of GLS1 as a therapeutic strategy to target mesenchymal DR PCa cells, thereby delaying accelerated tumour progression.

A reliable transcriptomic risk-score applicable to formalin-fixed paraffin-embedded biopsies improves outcome prediction in localized prostate cancer.

BACKGROUND: Clinical manifestation of prostate cancer (PCa) is highly variable. Aggressive tumors require radical treatment while clinically non-significant ones may be suitable for active surveillance. We previously developed the prognostic ProstaTrend RNA signature based on transcriptome-wide microarray and RNA-sequencing (RNA-Seq) analyses, primarily of prostatectomy specimens. An RNA-Seq study of formalin-fixed paraffin-embedded (FFPE) tumor biopsies has now allowed us to use this test as a basis for the development of a novel test that is applicable to FFPE biopsies as a tool for early routine PCa diagnostics. METHODS: All patients of the FFPE biopsy cohort were treated by radical prostatectomy and median follow-up for biochemical recurrence (BCR) was 9 years. Based on the transcriptome data of 176 FFPE biopsies, we filtered ProstaTrend for genes susceptible to FFPE-associated degradation via regression analysis. ProstaTrend was additionally restricted to genes with concordant prognostic effects in the RNA-Seq TCGA prostate adenocarcinoma (PRAD) cohort to ensure robust and broad applicability. The prognostic relevance of the refined Transcriptomic Risk Score (TRS) was analyzed by Kaplan-Meier curves and Cox-regression models in our FFPE-biopsy cohort and 9 other public datasets from PCa patients with BCR as primary endpoint. In addition, we developed a prostate single-cell atlas of 41 PCa patients from 5 publicly available studies to analyze gene expression of ProstaTrend genes in different cell compartments. RESULTS: Validation of the TRS using the original ProstaTrend signature in the cohort of FFPE biopsies revealed a relevant impact of FFPE-associated degradation on gene expression and consequently no significant association with prognosis (Cox-regression, p-value > 0.05) in FFPE tissue. However, the TRS based on the new version of the ProstaTrend-ffpe signature, which included 204 genes (of originally 1396 genes), was significantly associated with BCR in the FFPE biopsy cohort (Cox-regression p-value < 0.001) and retained prognostic relevance when adjusted for Gleason Grade Groups. We confirmed a significant association with BCR in 9 independent cohorts including 1109 patients. Comparison of the prognostic performance of the TRS with 17 other prognostically relevant PCa panels revealed that ProstaTrend-ffpe was among the best-ranked panels. We generated a PCa cell atlas to associate ProstaTrend genes with cell lineages or cell types. Tumor-specific luminal cells have a significantly higher TRS than normal luminal cells in all analyzed datasets. In addition, TRS of epithelial and luminal cells was correlated with increased Gleason score in 3 studies. CONCLUSIONS: We developed a prognostic gene-expression signature for PCa that can be applied to FFPE biopsies and may be suitable to support clinical decision-making.

The value of subcutaneous xenografts for individualised radiotherapy in HNSCC: Robust gene signature correlates with radiotherapy outcome in patients and xenografts.

PURPOSE: To assess the robustness of prognostic biomarkers and molecular tumour subtypes developed for patients with head and neck squamous cell carcinoma (HNSCC) on cell-line derived HNSCC xenograft models, and to develop a novel biomarker signature by combining xenograft and patient datasets. MATERIALS AND METHODS: Mice bearing xenografts (n = 59) of ten HNSCC cell lines and a retrospective, multicentre patient cohort (n = 242) of the German Cancer Consortium-Radiation Oncology Group (DKTK-ROG) were included. All patients received postoperative radiochemotherapy (PORT-C). Gene expression analysis was conducted using GeneChip Human Transcriptome Arrays. Xenografts were stratified based on their molecular subtypes and previously established gene classifiers. The dose to control 50 % of tumours (TCD50) was compared between these groups. Using differential gene expression analyses combining xenograft and patient data, a gene signature was developed to define risk groups for the primary endpoint loco-regional control (LRC). RESULTS: Tumours of mesenchymal subtype were characterized by a higher TCD50 (xenografts, p < 0.001) and lower LRC (patients, p < 0.001) compared to the other subtypes. Similar to previously published patient data, hypoxia- and radioresistance-related gene signatures were associated with high TCD50 values. A 2-gene signature (FN1, SERPINE1) was developed that was prognostic for TCD50 (xenografts, p < 0.001) and for patient outcome in independent validation (LRC: p = 0.007). CONCLUSION: Genetic prognosticators of outcome for patients after PORT-C and subcutaneous xenografts after primary clinically relevant irradiation show similarity. The identified robust 2-gene signature may help to guide patient stratification, after prospective validation. Thus, xenografts remain a valuable resource for translational research towards the development of individualized radiotherapy.

Standardized and quality-assured predictive PD-L1 testing in the upper gastrointestinal tract.

As a result of the high approval dynamics and the growing number of immuno-oncological concepts, the complexity of treatment decisions and control in the area of cancers of the esophagus, gastroesophageal junction and stomach is constantly increasing. Since the treatment indication for PD-1 inhibitors that are currently approved in the European Union is often linked to the expression of PD-L1 (programmed cell death-ligand 1), the evaluation of tissue-based predictive markers by the pathologist is of crucial importance for treatment stratification. Even though the immunohistochemical analysis of the PD-L1 expression status is one of the best studied, therapy-relevant biomarkers for an immuno-oncological treatment, due to the high heterogeneity of carcinomas of the upper gastrointestinal tract, there are challenges in daily clinical diagnostic work with regard to implementation, standardization and interpretation of testing. An interdisciplinary group of experts from Germany has taken a position on relevant questions from daily pathological and clinical practice, which concern the starting material, quality-assured testing and the interpretation of pathological findings, and has developed recommendations for structured reporting.

PD-L1 mRNA Detection in Immunohistochemically Negative Patients: A Complementary Method for a Better Treatment Selection?

BACKGROUND/AIM: PD-L1 inhibitors have been approved for cisplatin-ineligible urothelial cancer patients relapsing after radical cystectomy. A prerequisite for therapy is a positive PD-L1 expression in the tumor tissue, whereas no options are available for patients with negative PD-L1 status. However, studies revealed that many PD-L1-negative patients also responded to PD-L1 therapy. This study investigated the feasibility of PD-L1 mRNA complementary RNA in situ hybridization (RNAish) analysis to detect PD-L1-responders independent of PD-L1 protein status. MATERIALS AND METHODS: Immunohistochemistry and RNA in situ hybridization were used to assess PD-L1 protein and mRNA in radical cystectomy tissue from patients with advanced and metastasized urothelial cancer. RESULTS: In this study, PD-L1 protein and mRNA were detected in ≥90% of the examined tissue. Positive PD-L1 mRNA expression (≥1%) on TC and IC could be evaluated in 77% and 31% of the cases, respectively. Moreover, scatterplot analysis revealed a PD-L1 mRNA positive and PD-L1 protein negative subpopulation. According to the CPS score, positive PD-L1 protein expression could be evaluated in 88% and positive PD-L1 mRNA expression in 71% of the cases. Scatterplot analysis of the CPS scores revealed a CPS protein negative but CPS mRNA positive small subpopulation. CONCLUSION: The feasibility of RNAish on formalin-fixed tissue could be proven. Moreover, complementary PD-L1 RNAish identified a sub-population of PD-L1 protein-negative and PD-L1 mRNA-positive patients, which may benefit from PD-L1 therapy.

[Ulcerative colitis-associated carcinogenesis : An update]. / Colitis-ulcerosa-assoziierte Karzinogenese : Ein Update.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease beginning in the rectum and gradually extending to the right-sided colon and the terminal ileum (backwash-ileitis). Its causes are still not completely understood. Genetic susceptibility, changes in the microbiota and immune response, as well as environmental factors are thought to influence the disease course.Patients with UC are at increased risk of developing colorectal cancer (CRC) when compared to an age-matched normal population. Cancer risk increases with early onset, duration, and extent of the disease, with development of strictures, intraepithelial neoplasia, and concomitant primary sclerosing cholangitis.In contrast to the sporadic adenoma-carcinoma-sequence, UC-related CRC develops through an inflammation-intraepithelial neoplasia-carcinoma-sequence, in which genetic alterations already occur in the inflamed epithelium before the development of intraepithelial neoplasia.This article summarizes the current state of knowledge regarding UC-related carcinogenesis and its possible impact on prevention and therapy.

Characterization and evaluation of gene fusions as a measure of genetic instability and disease prognosis in prostate cancer.

BACKGROUND: Prostate cancer (PCa) is one of the most prevalent cancers worldwide. The clinical manifestations and molecular characteristics of PCa are highly variable. Aggressive types require radical treatment, whereas indolent ones may be suitable for active surveillance or organ-preserving focal therapies. Patient stratification by clinical or pathological risk categories still lacks sufficient precision. Incorporating molecular biomarkers, such as transcriptome-wide expression signatures, improves patient stratification but so far excludes chromosomal rearrangements. In this study, we investigated gene fusions in PCa, characterized potential novel candidates, and explored their role as prognostic markers for PCa progression. METHODS: We analyzed 630 patients in four cohorts with varying traits regarding sequencing protocols, sample conservation, and PCa risk group. The datasets included transcriptome-wide expression and matched clinical follow-up data to detect and characterize gene fusions in PCa. With the fusion calling software Arriba, we computationally predicted gene fusions. Following detection, we annotated the gene fusions using published databases for gene fusions in cancer. To relate the occurrence of gene fusions to Gleason Grading Groups and disease prognosis, we performed survival analyses using the Kaplan-Meier estimator, log-rank test, and Cox regression. RESULTS: Our analyses identified two potential novel gene fusions, MBTTPS2,L0XNC01::SMS and AMACR::AMACR. These fusions were detected in all four studied cohorts, providing compelling evidence for the validity of these fusions and their relevance in PCa. We also found that the number of gene fusions detected in a patient sample was significantly associated with the time to biochemical recurrence in two of the four cohorts (log-rank test, p-value < 0.05 for both cohorts). This was also confirmed after adjusting the prognostic model for Gleason Grading Groups (Cox regression, p-values < 0.05). CONCLUSIONS: Our gene fusion characterization workflow revealed two potential novel fusions specific for PCa. We found evidence that the number of gene fusions was associated with the prognosis of PCa. However, as the quantitative correlations were only moderately strong, further validation and assessment of clinical value is required before potential application.

Label-free multiphoton microscopy enables histopathological assessment of colorectal liver metastases and supports automated classification of neoplastic tissue.

As the state of resection margins is an important prognostic factor after extirpation of colorectal liver metastases, surgeons aim to obtain negative margins, sometimes elaborated by resections of the positive resection plane after intraoperative frozen sections. However, this is time consuming and results sometimes remain unclear during surgery. Label-free multimodal multiphoton microscopy (MPM) is an optical technique that retrieves morpho-chemical information avoiding all staining and that can potentially be performed in real-time. Here, we investigated colorectal liver metastases and hepatic tissue using a combination of three endogenous nonlinear signals, namely: coherent anti-Stokes Raman scattering (CARS) to visualize lipids, two-photon excited fluorescence (TPEF) to visualize cellular patterns, and second harmonic generation (SHG) to visualize collagen fibers. We acquired and analyzed over forty thousand MPM images of metastatic and normal liver tissue of 106 patients. The morphological information with biochemical specificity produced by MPM allowed discriminating normal liver from metastatic tissue and discerning the tumor borders on cryosections as well as formalin-fixed bulk tissue. Furthermore, automated tissue type classification with a correct rate close to 95% was possible using a simple approach based on discriminant analysis of texture parameters. Therefore, MPM has the potential to increase the precision of resection margins in hepatic surgery of metastases without prolonging surgical intervention.

Identification of Epigenetically Regulated Genes Distinguishing Intracranial from Extracranial Melanoma Metastases.

Despite remarkable advances in treating patients with metastatic melanoma, the management of melanoma brain metastases remains challenging. Recent evidence suggests that epigenetic reprogramming is an important mechanism for the adaptation of melanoma cells to the brain environment. In this study, the methylomes and transcriptomes of a cohort of matched melanoma metastases were evaluated by integrated omics data analysis. The identified 38 candidate genes displayed distinct promoter methylation and corresponding gene expression changes in intracranial compared with extracranial metastases. The 11 most promising genes were validated on protein level in both tumor and surrounding normal tissue using immunohistochemistry. In accordance with the underlying promoter methylation and gene expression changes, a significantly different protein expression was confirmed for STK10, PDXK, WDR24, CSSP1, NMB, RASL11B, phosphorylated PRKCZ, PRKCZ, and phosphorylated GRB10 in the intracranial metastases. The observed changes imply a distinct intracranial phenotype with increased protein kinase B phosphorylation and a higher frequency of proliferating cells. Knockdown of PRKCZ or GRB10 altered the expression of phosphorylated protein kinase B and decreased the viability of a brain-specific melanoma cell line. In summary, epigenetically regulated cancer-relevant alterations were identified that provide insights into the molecular mechanisms that discriminate brain metastases from other organ metastases, which could be exploited by targeting the affected signaling pathways.

Integrated radiogenomics analyses allow for subtype classification and improved outcome prognosis of patients with locally advanced HNSCC.

Patients with locally advanced head and neck squamous cell carcinoma (HNSCC) may benefit from personalised treatment, requiring biomarkers that characterize the tumour and predict treatment response. We integrate pre-treatment CT radiomics and whole-transcriptome data from a multicentre retrospective cohort of 206 patients with locally advanced HNSCC treated with primary radiochemotherapy to classify tumour molecular subtypes based on radiomics, develop surrogate radiomics signatures for gene-based signatures related to different biological tumour characteristics and evaluate the potential of combining radiomics features with full-transcriptome data for the prediction of loco-regional control (LRC). Using end-to-end machine-learning, we developed and validated a model to classify tumours of the atypical subtype (AUC [95% confidence interval] 0.69 [0.53-0.83]) based on CT imaging, observed that CT-based radiomics models have limited value as surrogates for six selected gene signatures (AUC < 0.60), and showed that combining a radiomics signature with a transcriptomics signature consisting of two metagenes representing the hedgehog pathway and E2F transcriptional targets improves the prognostic value for LRC compared to both individual sources (validation C-index [95% confidence interval], combined: 0.63 [0.55-0.73] vs radiomics: 0.60 [0.50-0.71] and transcriptomics: 0.59 [0.49-0.69]). These results underline the potential of multi-omics analyses to generate reliable biomarkers for future application in personalized oncology.

Influence of Androgen Deprivation Therapy on the PD-L1 Expression and Immune Activity in Prostate Cancer Tissue.

Immune checkpoint inhibitors have become a promising new therapy for cancer treatment. However, due to prostate cancer's high heterogeneity and immune-suppressive tumour microenvironment, clinical trials with immune checkpoint inhibitors for prostate cancer resulted in low or no response. This descriptive and retrospective study investigates the influence of androgen deprivation therapy (ADT) on PD-L1 expression and CD8+ T-cell tumour infiltration and activity in primary prostate cancer tissue. Therefore, immunohistochemistry was used to assess PD-L1, CD8+ T-cell, and the immune activation marker Granzyme B (GrB) in PCa tissue before and under ADT. In line with previous studies, few prostate cancer tissues showed PD-L1 expression and CD8+ T-cell infiltration. However, PD-L1 expression levels on tumour cells or infiltrating immune cells above 5% generated an immune-suppressive tumour microenvironment harbouring hypofunctional CD8+ T-cells. Moreover, analysis of a longitudinal patient cohort before and under ADT revealed that ADT increased hypofunctional CD8+ T cells in the tumour area suggesting a tumour immune milieu optimal for targeting with immunotherapy.

A Novel 2-Metagene Signature to Identify High-Risk HNSCC Patients amongst Those Who Are Clinically at Intermediate Risk and Are Treated with PORT.

(1) Background: Patients with locally advanced head and neck squamous cell carcinoma (HNSCC) who are biologically at high risk for the development of loco−regional recurrences after postoperative radiotherapy (PORT) but at intermediate risk according to clinical risk factors may benefit from additional concurrent chemotherapy. In this matched-pair study, we aimed to identify a corresponding predictive gene signature. (2) Methods: Gene expression analysis was performed on a multicenter retrospective cohort of 221 patients that were treated with postoperative radiochemotherapy (PORT-C) and 283 patients who were treated with PORT alone. Propensity score analysis was used to identify matched patient pairs from both cohorts. From differential gene expression analysis and Cox regression, a predictive gene signature was identified. (3) Results: 108 matched patient pairs were selected. We identified a 2-metagene signature that stratified patients into risk groups in both cohorts. The comparison of the high-risk patients between the two types of treatment showed higher loco−regional control (LRC) after treatment with PORT-C (p < 0.001), which was confirmed by a significant interaction term in Cox regression (p = 0.027), i.e., the 2-metagene signature was indicative for the type of treatment. (4) Conclusion: We have identified a novel gene signature that may be helpful to identify patients with high-risk HNSCC amongst those at intermediate clinical risk treated with PORT, who may benefit from additional concurrent chemotherapy.

Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer.

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.

Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancer.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.

Development and validation of a 6-gene signature for the prognosis of loco-regional control in patients with HPV-negative locally advanced HNSCC treated by postoperative radio(chemo)therapy.

PURPOSE: The aim of this study was to develop and validate a novel gene signature from full-transcriptome data using machine-learning approaches to predict loco-regional control (LRC) of patients with human papilloma virus (HPV)-negative locally advanced head and neck squamous cell carcinoma (HNSCC), who received postoperative radio(chemo)therapy (PORT-C). MATERIALS AND METHODS: Gene expression analysis was performed using Affymetrix GeneChip Human Transcriptome Array 2.0 on a multicentre retrospective training cohort of 128 patients and an independent validation cohort of 114 patients from the German Cancer Consortium - Radiation Oncology Group (DKTK-ROG). Genes were filtered based on differential gene expression analyses and Cox regression. The identified gene signature was combined with clinical parameters and with previously identified genes related to stem cells and hypoxia. Technical validation was performed using nanoString technology. RESULTS: We identified a 6-gene signature consisting of four individual genes CAV1, GPX8, IGLV3-25, TGFBI, and one metagene combining the highly correlated genes INHBA and SERPINE1. This signature was prognostic for LRC on the training data (ci = 0.84) and in validation (ci = 0.63) with a significant patient stratification into two risk groups (p = 0.005). Combining the 6-gene signature with the clinical parameters T stage and tumour localisation as well as the cancer stem cell marker CD44 and the 15-gene hypoxia-associated signature improved the validation performance (ci = 0.69, p = 0.001). CONCLUSION: We have developed and validated a novel prognostic 6-gene signature for LRC of HNSCC patients with HPV-negative tumours treated by PORT-C. After successful prospective validation the signature can be part of clinical trials on the individualization of radiotherapy.

Influence of Systemic Therapy on the Expression and Activity of Selected STAT Proteins in Prostate Cancer Tissue.

Signal Transducer and Activator of Transcription (STAT) proteins have been identified as drivers of prostate cancer (PCa) progression and development of aggressive castration-resistant phenotypes. In particular, STAT3, 5, and 6 have been linked to resistance to androgen receptor inhibition and metastasis in in vitro and in vivo models. This descriptive study aimed to validate these preclinical data in tissue obtained from patients with PCa before and while under androgen-deprivation therapy. Therefore, STAT3, 5, and 6 expressions and activity were assessed by immunohistochemistry. The data revealed that STAT3 and 5 changed in PCa. However, there was no relationship between expression and survival. Moreover, due to the heterogeneous nature of PCa, the preclinical results could not be transferred congruently to the patient's material. A pilot study with a longitudinal patient cohort could also show this heterogeneous influence of systemic therapy on STAT3, 5, and 6 expressions and activity. Even if the main mechanisms were validated, these data demonstrate the urge for better patient-near preclinical models. Therefore, these data reflect the need for investigations of STAT proteins in a longitudinal patient cohort to identify factors responsible for the diverse influence of system therapy on STAT expression.

Biomarker signatures for primary radiochemotherapy of locally advanced HNSCC - Hypothesis generation on a multicentre cohort of the DKTK-ROG.

PURPOSE: To develop prognostic biomarker signatures for patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated by primary radiochemotherapy (RCTx) based on previously published molecular analyses of the retrospective biomarker study of the German Cancer Consortium - Radiation Oncology Group (DKTK-ROG). MATERIAL AND METHODS: In previous studies on the retrospective DKTK-ROG HNSCC cohort treated with primary RCTx, the following clinical parameters and biomarkers were evaluated and found to be significantly associated with loco-regional tumour control (LRC) or overall survival (OS): tumour volume, p16 status, expression of cancer stem cell markers CD44 and SLC3A2, expressions of hypoxia-associated gene signatures, tumour mutational burden (TMB), single nucleotide polymorphisms (SNPs) in the ERCC2 gene (rs1799793, rs13181) and ERCC5 gene (rs17655) as well as the expression of CXCR4, SDF-1 and CD8. These biomarkers were combined in multivariable modelling using Cox-regression with backward variable selection. RESULTS: A baseline signature containing the widely accepted parameters tumour volume, p16 status, cancer stem cell marker expression (CD44), and hypoxia-associated gene expression has been defined, representing the main hypothesis of the study. Furthermore, the baseline signature was extended by additional prognostic biomarkers and a data-driven signature without any pre-hypothesis was generated for both endpoints. In these signatures, the SNPs rs1799793 and rs17655 as well as CXCR4, SDF-1 and SLC3A2 expression were additionally included. The signatures showed significant patient stratifications for LRC and OS. CONCLUSION: Three biomarker signatures were defined for patients with locally advanced HNSCC treated with primary RCTx for the endpoints LRC and OS. These signatures will be validated in the prospective HNprädBio study of the DKTK-ROG that recently completed recruitment, before potential application in an interventional trial.

Impact of Androgen Receptor Activity on Prostate-Specific Membrane Antigen Expression in Prostate Cancer Cells.

Prostate-specific membrane antigen (PSMA) is an essential molecular regulator of prostate cancer (PCa) progression coded by the FOLH1 gene. The PSMA protein has become an important factor in metastatic PCa diagnosis and radioligand therapy. However, low PSMA expression is suggested to be a resistance mechanism to PSMA-based imaging and therapy. Clinical studies revealed that androgen receptor (AR) inhibition increases PSMA expression. The mechanism has not yet been elucidated. Therefore, this study investigated the effect of activation and inhibition of androgen signaling on PSMA expression levels in vitro and compared these findings with PSMA levels in PCa patients receiving systemic therapy. To this end, LAPC4, LNCaP, and C4-2 PCa cells were treated with various concentrations of the synthetic androgen R1881 and antiandrogens. Changes in FOLH1 mRNA were determined using qPCR. Open access databases were used for ChIP-Seq and tissue expression analysis. Changes in PSMA protein were determined using western blot. For PSMA staining in patients' specimens, immunohistochemistry (IHC) was performed. Results revealed that treatment with the synthetic androgen R1881 led to decreased FOLH1 mRNA and PSMA protein. This effect was partially reversed by antiandrogen treatment. However, AR ChIP-Seq analysis revealed no canonical AR binding sites in the regulatory elements of the FOLH1 gene. IHC analysis indicated that androgen deprivation only resulted in increased PSMA expression in patients with low PSMA levels. The data demonstrate that AR activation and inhibition affects PSMA protein levels via a possible non-canonical mechanism. Moreover, analysis of PCa tissue reveals that low PSMA expression rates may be mandatory to increase PSMA by androgen deprivation.

Analyses of molecular subtypes and their association to mechanisms of radioresistance in patients with HPV-negative HNSCC treated by postoperative radiochemotherapy.

PURPOSE: To assess the relation of the previously reported classification of molecular subtypes to the outcome of patients with HNSCC treated with postoperative radio(chemo)therapy (PORT-C), and to assess the association of these subtypes with gene expressions reflecting known mechanisms of radioresistance. MATERIAL AND METHODS: Gene expression analyses were performed using the GeneChip Human Transcriptome Array 2.0 on a multicentre retrospective patient cohort (N = 128) of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG) with locally advanced HNSCC treated with PORT-C. Tumours were assigned to four molecular subtypes, and correlation analyses between subtypes and clinical risk factors were performed. In addition, the classifications of eight genes or gene signatures related to mechanisms of radioresistance, which have previously shown an association with outcome of patients with HNSCC, were compared between the molecular subtypes. The endpoints loco-regional control (LRC) and overall survival (OS) were evaluated by log-rank tests and Cox regression. RESULTS: Tumours were classified into the four subtypes basal (19.5%), mesenchymal (18.8%), atypical (15.6%) and classical (14.1%). The remaining tumours could not be classified (32.0%). Tumours of the mesenchymal subtype showed a lower LRC compared to the other subtypes (p = 0.012). These tumours were associated with increased epithelial-mesenchymal transition (EMT) and overexpression of a gene signature enriched in DNA repair genes. The majority of the eight considered gene classifiers were significantly associated to LRC or OS in the whole cohort. CONCLUSION: Molecular subtypes, previously identified on HNSCC patients treated with primary radio(chemo)therapy or surgery, were related to LRC for patients treated with PORT-C, where mesenchymal tumours presented with worse prognosis. After prospective validation, subtype-based patient stratification, potentially in combination with other molecular classifiers, may be considered in future interventional studies in the context of personalised radiotherapy and may guide the development of combined treatment approaches.

Macroscopic, histologic, and clinical assessment of acute graft-versus-host disease of the upper gastrointestinal tract within 6 weeks after allogeneic hematopoietic cell transplantation.

Acute graft-versus-host-disease (aGVHD) is the main cause of morbidity and nonrelapse mortality (NRM) following allogeneic hematopoietic cell transplantation (alloHCT). Nausea, vomiting, and anorexia after alloHCT can be early signs of aGVHD of the gastrointestinal tract (GIT) but may also reflect lasting mucosal damage or side effects of drugs. If upper GIT aGVHD is suspected, upper endoscopic evaluation and histological examination are crucial. Still, the interpretation of clinical symptoms, macroscopical alterations, and histological findings can be challenging. Therefore, we conducted a retrospective analysis on single-center data from 174 patients with suspected aGVHD of the upper GIT who underwent upper endoscopy within the first 6 weeks after alloHCT, to study the distribution of aGVHD-related histological findings in relation to clinical symptoms and macroscopic findings and to correlate the severity of changes with data on relapse and NRM. Our data suggest that biopsies of the duodenum reveal the severity of upper GIT aGVHD most accurately. While the histological grading correlated weakly with the severity of macroscopic changes, we found a tight correlation between histological and clinical grades of upper GIT aGVHD (p < 0.001). Although correlation of histological grading of upper GIT aGVHD with the risk for NRM missed statistical significance (HR 1.53, Lerner ≥1° versus <1º, p = 0.13), overall clinical aGVHD severity correlated with NRM (HR 4.3, IIIº-IVº versus 0-Iº, p < 0.01). In conclusion, biopsies from the duodenum are most sensitive in excluding aGVHD in patients with normal macroscopic findings at esophagogastroduodenoscopy. Clinical grading of aGVHD predicts NRM better than histological grading.