Automated Evaluation of p16/Ki-67 Dual-Stain Cytology as a Biomarker for Detection of Anal Precancer in Men Who Have Sex With Men and Are Living With Human Immunodeficiency Virus.

BACKGROUND: Human papillomavirus-related biomarkers such as p16/Ki-67 "dual-stain" (DS) cytology have shown promising clinical performance for anal cancer screening. Here, we assessed the performance of automated evaluation of DS cytology (automated DS) to detect anal precancer in men who have sex with men (MSM) and are living with human immunodeficiency virus (HIV). METHODS: We conducted a cross-sectional analysis of 320 MSM with HIV undergoing anal cancer screening and high-resolution anoscopy (HRA) in 2009-2010. We evaluated the performance of automated DS based on a deep-learning classifier compared to manual evaluation of DS cytology (manual DS) to detect anal intraepithelial neoplasia grade 2 or 3 (AIN2+) and grade 3 (AIN3). We evaluated different DS-positive cell thresholds quantified by the automated approach and modeled performance compared with other screening strategies in a hypothetical population of MSM with HIV. RESULTS: Compared with manual DS, automated DS had significantly higher specificity (50.9% vs 42.2%; P < .001) and similar sensitivity (93.2% vs 92.1%) for detection of AIN2+. Human papillomavirus testing with automated DS triage was significantly more specific than automated DS alone (56.5% vs 50.9%; P < .001), with the same sensitivity (93.2%). In a modeled analysis assuming a 20% AIN2+ prevalence, automated DS detected more precancers than manual DS and anal cytology (186, 184, and 162, respectively) and had the lowest HRA referral rate per AIN2+ case detected (3.1, 3.5, and 3.3, respectively). CONCLUSIONS: Compared with manual DS, automated DS detects the same number of precancers, with a lower HRA referral rate.

The impact of SAMHD1 expression and mutation status in mantle cell lymphoma: An analysis of the MCL Younger and Elderly trial.

The sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) has been demonstrated to predict the response to high-dose cytarabine consolidation treatment in acute myeloid leukemia patients. Here, we evaluated SAMHD1 as potential biomarker for the response to high-dose cytarabine in mantle cell lymphoma (MCL) patients. We quantified SAMHD1 protein expression and determined the mutation status in patients of the MCL Younger and Elderly trials (n = 189), who had received high-dose cytarabine- or fludarabine-based polychemotherapy. Additionally, we quantified SAMHD1 expression in B cell lymphoma cell lines and exposed them to cytarabine, fludarabine, and clinically relevant combinations. Across both trials investigated, SAMHD1 mutations had a frequency of 7.1% (n = 13) and did not significantly affect the failure-free survival (FFS, P = .47). In patients treated with high-dose cytarabine- or fludarabine-containing regimes, SAMHD1 expression was not significantly associated with FFS or complete remission rate. SAMHD1 expression in B cell lymphoma cell lines, however, inversely correlated with their in vitro response to cytarabine as single agent (R = .65, P = .0065). This correlation could be reversed by combining cytarabine with other chemotherapeutics, such as oxaliplatin and vincristine, similar to the treatment regime of the MCL Younger trial. We conclude that this might explain why we did not observe a significant association between SAMHD1 protein expression and the outcome of MCL patients upon cytarabine-based treatment.

Co-expression of YAP and TAZ associates with chromosomal instability in human cholangiocarcinoma.

BACKGROUND: Activation of the oncogene yes-associated protein (YAP) is frequently detected in intrahepatic cholangiocarcinoma (iCCA); however, the expression pattern and the functional impact of its paralogue WW domain-containing transcription regulator 1 (WWTR1; synonym: TAZ) are not well described in different CCA subtypes. METHODS: Immunohistochemical analysis of YAP and TAZ in iCCA and extrahepatic CCA (eCCA) cohorts was performed. YAP/TAZ shuttling and their functional impact on CCA cell lines were investigated. Target genes expression after combined YAP/TAZ inhibition was analyzed. RESULTS: Immunohistochemical analysis of iCCA and eCCA revealed YAP or TAZ positivity in up to 49.2%; however, oncogene co-expression was less frequent (up to 23%). In contrast, both proteins were jointly detectable in most CCA cell lines and showed nuclear/cytoplasmic shuttling in a cell density-dependent manner. Next to the pro-proliferative function of YAP/TAZ, both transcriptional co-activators cooperated in the regulation of a gene signature that indicated the presence of chromosomal instability (CIN). A correlation between YAP and the CIN marker phospho-H2A histone family member X (pH2AX) was particularly observed in tissues from iCCA and distal CCA (dCCA). The presence of the CIN genes in about 25% of iCCA was statistically associated with worse prognosis. CONCLUSIONS: YAP and TAZ activation is not uncoupled from cell density in CCA cells and both factors cooperatively contribute to proliferation and expression of CIN-associated genes. The corresponding group of CCA patients is characterized by CIN and may benefit from YAP/TAZ-directed therapies.

Identification and regulation of the long non-coding RNA Heat2 in heart failure.

AIMS: Circulating immune cells have a significant impact on progression and outcome of heart failure. Long non-coding RNAs (lncRNAs) comprise novel epigenetic regulators which control cardiovascular diseases and inflammatory disorders. We aimed to identify lncRNAs regulated in circulating immune cells of the blood of heart failure patients. METHODS AND RESULTS: Next-generation sequencing revealed 110 potentially non-coding RNA transcripts differentially expressed in peripheral blood mononuclear cells of heart failure patients with reduced ejection fraction. The up-regulated lncRNA Heat2 was further functionally characterized. Heat2 expression was detected in whole blood, PBMNCs, eosinophil and basophil granulocytes. Heat2 regulates cell division, invasion, transmigration and immune cell adhesion on endothelial cells. CONCLUSION: Heat2 is an immune cell enriched lncRNA that is elevated in the blood of heart failure patients and controls cellular functions.

High endothelial venules are associated with microsatellite instability, hereditary background and immune evasion in colorectal cancer.

BACKGROUND: Microsatellite-unstable (MSI) tumours show a high load of mutational neoantigens, as a consequence of DNA mismatch repair deficiency. Consequently, MSI tumours commonly present with dense immune infiltration and develop immune evasion mechanisms. Whether improved lymphocyte recruitment contributes to the pronounced immune infiltration in MSI tumours is unknown. We analysed the density of high endothelial venules (HEV) and postcapillary blood vessels specialised for lymphocyte trafficking, in MSI colorectal cancers (CRC). METHODS: HEV density was determined by immunohistochemical staining of FFPE tissue sections from MSI (n = 48) and microsatellite-stable (MSS, n = 35) CRCs. Associations with clinical and pathological variables were analysed. RESULTS: We found elevated HEV densities in MSI compared with MSS CRCs (median 0.049 vs 0.000 counts/mm2, respectively, p = 0.0002), with the highest densities in Lynch syndrome MSI CRCs. Dramatically elevated HEV densities were observed in B2M-mutant Lynch syndrome CRCs, pointing towards a link between lymphocyte recruitment and immune evasion (median 0.485 vs 0.0885 counts/mm2 in B2M-wild-type tumours, p = 0.0237). CONCLUSIONS: Our findings for the first time indicate a significant contribution of lymphocyte trafficking in immune responses against MSI CRC, particularly in the context of Lynch syndrome. High HEV densities in B2M-mutant tumours underline the significance of immunoediting during tumour evolution.

Astrocytic glutamine synthetase is expressed in the neuronal somatic layers and down-regulated proportionally to neuronal loss in the human epileptic hippocampus.

Human mesial temporal lobe epilepsy (MTLE) features subregion-specific hippocampal neurodegeneration and reactive astrogliosis, including up-regulation of the glial fibrillary acidic protein (GFAP) and down-regulation of glutamine synthetase (GS). However, the regional astrocytic expression pattern of GFAP and GS upon MTLE-associated neurodegeneration still remains elusive. We assessed GFAP and GS expression in strict correlation with the local neuronal number in cortical and hippocampal surgical specimens from 16 MTLE patients using immunohistochemistry, stereology and high-resolution image analysis for digital pathology and whole-slide imaging. In the cortex, GS-positive (GS+) astrocytes are dominant in all neuronal layers, with a neuron to GS+ cell ratio of 2:1. GFAP-positive (GFAP+) cells are widely spaced, with a GS+ to GFAP+ cell ratio of 3:1-5:1. White matter astrocytes, on the contrary, express mainly GFAP and, to a lesser extent, GS. In the hippocampus, the neuron to GS+ cell ratio is approximately 1:1. Hippocampal degeneration is associated with a reduction of GS+ astrocytes, which is proportional to the degree of neuronal loss and primarily present in the hilus. Up-regulation of GFAP as a classical hallmark of reactive astrogliosis does not follow the GS-pattern and is prominent in the CA1. Reactive alterations were proportional to the neuronal loss in the neuronal somatic layers (stratum pyramidale and hilus), while observed to a lesser extent in the axonal/dendritic layers (stratum radiatum, molecular layer). We conclude that astrocytic GS is expressed in the neuronal somatic layers and, upon neurodegeneration, is down-regulated proportionally to the degree of neuronal loss.

Skin Cancer Classification Using Convolutional Neural Networks: Systematic Review.

BACKGROUND: State-of-the-art classifiers based on convolutional neural networks (CNNs) were shown to classify images of skin cancer on par with dermatologists and could enable lifesaving and fast diagnoses, even outside the hospital via installation of apps on mobile devices. To our knowledge, at present there is no review of the current work in this research area. OBJECTIVE: This study presents the first systematic review of the state-of-the-art research on classifying skin lesions with CNNs. We limit our review to skin lesion classifiers. In particular, methods that apply a CNN only for segmentation or for the classification of dermoscopic patterns are not considered here. Furthermore, this study discusses why the comparability of the presented procedures is very difficult and which challenges must be addressed in the future. METHODS: We searched the Google Scholar, PubMed, Medline, ScienceDirect, and Web of Science databases for systematic reviews and original research articles published in English. Only papers that reported sufficient scientific proceedings are included in this review. RESULTS: We found 13 papers that classified skin lesions using CNNs. In principle, classification methods can be differentiated according to three principles. Approaches that use a CNN already trained by means of another large dataset and then optimize its parameters to the classification of skin lesions are the most common ones used and they display the best performance with the currently available limited datasets. CONCLUSIONS: CNNs display a high performance as state-of-the-art skin lesion classifiers. Unfortunately, it is difficult to compare different classification methods because some approaches use nonpublic datasets for training and/or testing, thereby making reproducibility difficult. Future publications should use publicly available benchmarks and fully disclose methods used for training to allow comparability.

Teledermatology: Comparison of Store-and-Forward Versus Live Interactive Video Conferencing.

A decreasing number of dermatologists and an increasing number of patients in Western countries have led to a relative lack of clinicians providing expert dermatologic care. This, in turn, has prolonged wait times for patients to be examined, putting them at risk. Store-and-forward teledermatology improves patient access to dermatologists through asynchronous consultations, reducing wait times to obtain a consultation. However, live video conferencing as a synchronous service is also frequently used by practitioners because it allows immediate interaction between patient and physician. This raises the question of which of the two approaches is superior in terms of quality of care and convenience. There are pros and cons for each in terms of technical requirements and features. This viewpoint compares the two techniques based on a literature review and a clinical perspective to help dermatologists assess the value of teledermatology and determine which techniques would be valuable in their practice.

Identification of T cell target antigens in glioblastoma stem-like cells using an integrated proteomics-based approach in patient specimens.

Glioblastoma (GBM) is a highly aggressive brain tumor and still remains incurable. Among others, an immature subpopulation of self-renewing and therapy-resistant tumor cells-often referred to as glioblastoma stem-like cells (GSCs)-has been shown to contribute to disease recurrence. To target these cells personalized immunotherapy has gained a lot of interest, e.g. by reactivating pre-existing anti-tumor immune responses against GSC antigens. To identify T cell targets commonly presented by GSCs and their differentiated counterpart, we used a proteomics-based separation of GSC proteins in combination with a T cell activation assay. Altogether, 713 proteins were identified by LC-ESI-MS/MS mass spectrometry. After a thorough filtering process, 32 proteins were chosen for further analyses. Immunogenicity of corresponding peptides was tested ex vivo. A considerable number of these antigens induced T cell responses in GBM patients but not in healthy donors. Moreover, most of them were overexpressed in primary GBM and also highly expressed in recurrent GBM tissues. Interestingly, expression of the most frequent T cell target antigens could also be confirmed in quiescent, slow-cycling GSCs isolated in high purity by the DEPArray technology. Finally, for a subset of these T cell target antigens, an association between expression levels and higher T cell infiltration as well as an increased expression of positive immune modulators was observed. In summary, we identified novel immunogenic proteins, which frequently induce tumor-specific T cell responses in GBM patients and were also detected in vitro in therapy-resistant quiescent, slow-cycling GSCs. Stable expression of these T cell targets in primary and recurrent GBM support their suitability for future clinical use.

Glyoxalase 1 expression is associated with an unfavorable prognosis of oropharyngeal squamous cell carcinoma.

BACKGROUND: Glyoxalase 1 is a key enzyme in the detoxification of reactive metabolites such as methylglyoxal and induced Glyoxalase 1 expression has been demonstrated for several human malignancies. However, the regulation and clinical relevance of Glyoxalase 1 in the context of head and neck squamous cell carcinoma has not been addressed so far. METHODS: Argpyrimidine modification as a surrogate for methylglyoxal accumulation and Glyoxalase 1 expression in tumor cells was assessed by immunohistochemical staining of tissue microarrays with specimens from oropharyngeal squamous cell carcinoma patients (n = 154). Prognostic values of distinct Glyoxalase 1 staining patterns were demonstrated by Kaplan-Meier, univariate and multivariate Cox proportional hazard model analysis. The impact of exogenous methylglyoxal or a Glyoxalase 1 inhibitor on the viability of two established tumor cell lines was monitored by a colony-forming assay in vitro. RESULTS: Glyoxalase 1 expression in tumor cells of oropharyngeal squamous cell carcinoma patients was positively correlated with the presence of Argpyrimidine modification and administration of exogenous methylglyoxal induced Glyoxalase 1 protein levels in FaDu and Cal27 cells in vitro. Cal27 cells with lower basal and methylglyoxal-induced Glyoxalase 1 expression were more sensitive to the cytotoxic effect at high methylgyoxal concentrations and both cell lines showed a decrease in colony formation with increasing amounts of a Glyoxalase 1 inhibitor. A high and nuclear Glyoxalase 1 staining was significantly correlated with shorter progression-free and disease-specific survival, and served as an independent risk factor for an unfavorable prognosis of oropharyngeal squamous cell carcinoma patients. CONCLUSIONS: Induced Glyoxalase 1 expression is a common feature in the pathogenesis of oropharyngeal squamous cell carcinoma and most likely represents an adaptive response to the accumulation of cytotoxic metabolites. Oropharyngeal squamous cell carcinoma patients with a high and nuclear Glyoxalase 1 staining pattern have a high risk for treatment failure, but might benefit from pharmacological targeting Glyoxalase 1 activity.

LOC283731 promoter hypermethylation prognosticates survival after radiochemotherapy in IDH1 wild-type glioblastoma patients.

MGMT promoter methylation status is currently the only established molecular prognosticator in IDH wild-type glioblastoma multiforme (GBM). Therefore, we aimed to discover novel therapy-associated epigenetic biomarkers. After enrichment for hypermethylated fractions using methyl-CpG-immunoprecipitation (MCIp), we performed global DNA methylation profiling for 14 long-term (LTS; >36 months) and 15 short-term (STS; 6-10 months) surviving GBM patients. Even after exclusion of the G-CIMP phenotype, we observed marked differences between the LTS and STS methylome. A total of 1,247 probes in 706 genes were hypermethylated in LTS and 463 probes in 305 genes were found to be hypermethylated in STS patients (p values < 0.05, log2 fold change ± 0.5). We identified 13 differentially methylated regions (DMRs) with a minimum of four differentially methylated probes per gene. Indeed, we were able to validate a subset of these DMRs through a second, independent method (MassARRAY) in our LTS/STS training set (ADCY1, GPC3, LOC283731/ISLR2). These DMRs were further assessed for their prognostic capability in an independent validation cohort (n = 62) of non-G-CIMP GBMs from the TCGA. Hypermethylation of multiple CpGs mapping to the promoter region of LOC283731 correlated with improved patient outcome (p = 0.03). The prognostic performance of LOC283731 promoter hypermethylation was confirmed in a third independent study cohort (n = 89), and was independent of gender, performance (KPS) and MGMT status (p = 0.0485, HR = 0.63). Intriguingly, the prediction was most pronounced in younger GBM patients (<60 years). In conclusion, we provide compelling evidence that promoter methylation status of this novel gene is a prognostic biomarker in IDH1 wild-type/non-G-CIMP GBMs.

Spatial transcriptome analysis reveals Notch pathway-associated prognostic markers in IDH1 wild-type glioblastoma involving the subventricular zone.

BACKGROUND: The spatial relationship of glioblastoma (GBM) to the subventricular zone (SVZ) is associated with inferior patient survival. However, the underlying molecular phenotype is largely unknown. We interrogated an SVZ-dependent transcriptome and potential location-specific prognostic markers. METHODS: mRNA microarray data of a discovery set (n = 36 GBMs) were analyzed for SVZ-dependent gene expression and process networks using the MetaCore™ workflow. Differential gene expression was confirmed by qPCR in a validation set of 142 IDH1 wild-type GBMs that was also used for survival analysis. RESULTS: Microarray analysis revealed a transcriptome distinctive of SVZ+ GBM that was enriched for genes associated with Notch signaling. No overlap was found to The Cancer Genome Atlas's molecular subtypes. Independent validation of SVZ-dependent expression confirmed four genes with simultaneous prognostic impact: overexpression of HES4 (p = 0.034; HR 1.55) and DLL3 (p = 0.017; HR 1.61) predicted inferior, and overexpression of NTRK2 (p = 0.049; HR 0.66) and PIR (p = 0.025; HR 0.62) superior overall survival (OS). Additionally, overexpression of DLL3 was predictive of shorter progression-free survival (PFS) (p = 0.043; HR 1.64). Multivariate analysis revealed overexpression of HES4 to be independently associated with inferior OS (p = 0.033; HR 2.03), and overexpression of DLL3 with inferior PFS (p = 0.046; HR 1.65). CONCLUSIONS: We identified four genes with SVZ-dependent expression and prognostic significance, among those HES4 and DLL3 as part of Notch signaling, suggesting further evaluation of location-tailored targeted therapies.

Oral treatment with a zinc complex of acetylsalicylic acid prevents diabetic cardiomyopathy in a rat model of type-2 diabetes: activation of the Akt pathway.

BACKGROUND: Type-2 diabetics have an increased risk of cardiomyopathy, and heart failure is a major cause of death among these patients. Growing evidence indicates that proinflammatory cytokines may induce the development of insulin resistance, and that anti-inflammatory medications may reverse this process. We investigated the effects of the oral administration of zinc and acetylsalicylic acid, in the form of bis(aspirinato)zinc(II)-complex Zn(ASA)2, on different aspects of cardiac damage in Zucker diabetic fatty (ZDF) rats, an experimental model of type-2 diabetic cardiomyopathy. METHODS: Nondiabetic control (ZL) and ZDF rats were treated orally with vehicle or Zn(ASA)2 for 24 days. At the age of 29-30 weeks, the electrical activities, left-ventricular functional parameters and left-ventricular wall thicknesses were assessed. Nitrotyrosine immunohistochemistry, TUNEL-assay, and hematoxylin-eosin staining were performed. The protein expression of the insulin-receptor and PI3K/AKT pathway were quantified by Western blot. RESULTS: Zn(ASA)2-treatment significantly decreased plasma glucose concentration in ZDF rats (39.0 ± 3.6 vs 49.4 ± 2.8 mM, P < 0.05) while serum insulin-levels were similar among the groups. Data from cardiac catheterization showed that Zn(ASA)2 normalized the increased left-ventricular diastolic stiffness (end-diastolic pressure-volume relationship: 0.064 ± 0.008 vs 0.084 ± 0.014 mmHg/µl; end-diastolic pressure: 6.5 ± 0.6 vs 7.9 ± 0.7 mmHg, P < 0.05). Furthermore, ECG-recordings revealed a restoration of prolonged QT-intervals (63 ± 3 vs 83 ± 4 ms, P < 0.05) with Zn(ASA)2. Left-ventricular wall thickness, assessed by echocardiography, did not differ among the groups. However histological examination revealed an increase in the cardiomyocytes' transverse cross-section area in ZDF compared to the ZL rats, which was significantly decreased after Zn(ASA)2-treatment. Additionally, a significant fibrotic remodeling was observed in the diabetic rats compared to ZL rats, and Zn(ASA)2-administered ZDF rats showed a similar collagen content as ZL animals. In diabetic hearts Zn(ASA)2 significantly decreased DNA-fragmentation, and nitro-oxidative stress, and up-regulated myocardial phosphorylated-AKT/AKT protein expression. Zn(ASA)2 reduced cardiomyocyte death in a cellular model of oxidative stress. Zn(ASA)2 had no effects on altered myocardial CD36, GLUT-4, and PI3K protein expression. CONCLUSIONS: We demonstrated that treatment of type-2 diabetic rats with Zn(ASA)2 reduced plasma glucose-levels and prevented diabetic cardiomyopathy. The increased myocardial AKT activation could, in part, help to explain the cardioprotective effects of Zn(ASA)2. The oral administration of Zn(ASA)2 may have therapeutic potential, aiming to prevent/treat cardiac complications in type-2 diabetic patients.

Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients.

BACKGROUND: Dysregulated expression of Kallikrein-related peptidase 6 (KLK6) is a common feature for many human malignancies and numerous studies evaluated KLK6 as a promising biomarker for early diagnosis or unfavorable prognosis. However, the expression of KLK6 in carcinomas derived from mucosal epithelia, including head and neck squamous cell carcinoma (HNSCC), and its mode of action has not been addressed so far. METHODS: Stable clones of human mucosal tumor cell lines were generated with shRNA-mediated silencing or ectopic overexpression to characterize the impact of KLK6 on tumor relevant processes in vitro. Tissue microarrays with primary HNSCC samples from a retrospective patient cohort (n = 162) were stained by immunohistochemistry and the correlation between KLK6 staining and survival was addressed by univariate Kaplan-Meier and multivariate Cox proportional hazard model analysis. RESULTS: KLK6 expression was detected in head and neck tumor cell lines (FaDu, Cal27 and SCC25), but not in HeLa cervix carcinoma cells. Silencing in FaDu cells and ectopic expression in HeLa cells unraveled an inhibitory function of KLK6 on tumor cell proliferation and mobility. FaDu clones with silenced KLK6 expression displayed molecular features resembling epithelial-to-mesenchymal transition, nuclear ß-catenin accumulation and higher resistance against irradiation. Low KLK6 protein expression in primary tumors from oropharyngeal and laryngeal SCC patients was significantly correlated with poor progression-free (p = 0.001) and overall survival (p < 0.0005), and served as an independent risk factor for unfavorable clinical outcome. CONCLUSIONS: In summary, detection of low KLK6 expression in primary tumors represents a promising tool to stratify HNSCC patients with high risk for treatment failure. These patients might benefit from restoration of KLK6 expression or pharmacological targeting of signaling pathways implicated in EMT.

Impaired aldehyde dehydrogenase 1 subfamily member 2A-dependent retinoic acid signaling is related with a mesenchymal-like phenotype and an unfavorable prognosis of head and neck squamous cell carcinoma.

BACKGROUND: An inverse correlation between expression of the aldehyde dehydrogenase 1 subfamily A2 (ALDH1A2) and gene promoter methylation has been identified as a common feature of oropharyngeal squamous cell carcinoma (OPSCC). Moreover, low ALDH1A2 expression was associated with an unfavorable prognosis of OPSCC patients, however the causal link between reduced ALDH1A2 function and treatment failure has not been addressed so far. METHODS: Serial sections from tissue microarrays of patients with primary OPSCC (n = 101) were stained by immunohistochemistry for key regulators of retinoic acid (RA) signaling, including ALDH1A2. Survival with respect to these regulators was investigated by univariate Kaplan-Meier analysis and multivariate Cox regression proportional hazard models. The impact of ALDH1A2-RAR signaling on tumor-relevant processes was addressed in established tumor cell lines and in an orthotopic mouse xenograft model. RESULTS: Immunohistochemical analysis showed an improved prognosis of ALDH1A2(high) OPSCC only in the presence of CRABP2, an intracellular RA transporter. Moreover, an ALDH1A2(high)CRABP2(high) staining pattern served as an independent predictor for progression-free (HR: 0.395, p = 0.007) and overall survival (HR: 0.303, p = 0.002), suggesting a critical impact of RA metabolism and signaling on clinical outcome. Functionally, ALDH1A2 expression and activity in tumor cell lines were related to RA levels. While administration of retinoids inhibited clonogenic growth and proliferation, the pharmacological inhibition of ALDH1A2-RAR signaling resulted in loss of cell-cell adhesion and a mesenchymal-like phenotype. Xenograft tumors derived from FaDu cells with stable silencing of ALDH1A2 and primary tumors from OPSCC patients with low ALDH1A2 expression exhibited a mesenchymal-like phenotype characterized by vimentin expression. CONCLUSIONS: This study has unraveled a critical role of ALDH1A2-RAR signaling in the pathogenesis of head and neck cancer and our data implicate that patients with ALDH1A2(low) tumors might benefit from adjuvant treatment with retinoids.

High S100A8 and S100A12 protein expression is a favorable prognostic factor for survival of oropharyngeal squamous cell carcinoma.

S100/calgranulins (S100A8, S100A9 and S100A12) are key players of innate immune function and elevated levels are a characteristic feature of acute and chronic inflammation, and inflammation-associated carcinogenesis. However, reduced S100A8 and S100A9 expression has been detected for squamous cell carcinoma, including the head and neck region (HNSCC), which originate from mucosal epithelia with abundant expression of both proteins under physiological conditions. In contrast to S100A8 and S100A9, only sparse information is available for S100A12 and a comparative study of all three S100/calgranulins in HNSCC is still missing. We analyzed S100/calgranulin protein levels in a retrospective patient cohort (n = 131) of oropharyngeal squamous cell carcinoma (OPSCC) by immunohistochemical staining of tissue microarrays. Common characteristics of all three S100/calgranulins were: (i) abundant expression in supra-basal keratinocytes of normal mucosa with predominant nuclear staining, (ii) low expression in 30.4-51.9% of primary OPSCCs and (iii) variable accumulation of S100/calgranulin-positive immune cells in the tumor stroma. These features were associated with histopathological characteristics, such as tumor grade, lymph node metastasis and tumor stage. Furthermore, univariate and multivariate analysis revealed worse overall survival of OPSCC patients with simultaneous reduction of S100A8 and S100A12 expression, while expression of S100A9 or presence of the S100A8/S100A9 heterodimer had no impact, suggesting distinct regulation and function of individual S100/calgranulins in the pathogenesis of HNSCCs.

Phosphorylation of AKT(Ser473) serves as an independent prognostic marker for radiosensitivity in advanced head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by high resistance to radiotherapy, which critically depends on both altered signaling pathways within tumor cells and their dynamic interaction with the tumor microenvironment. This study evaluated the prognostic value of the phosphorylation status of AKT on Ser473 and Thr308 for the clinical outcome of patients with advanced HNSCC on radiotherapy. Furthermore, we investigated the impact of AKT(Ser473) phosphorylation [p-AKT(Ser473)] in the context of radioresistance using ex vivo tissue cultures that resemble the complex tissue architecture and paracrine interaction with the tumor microenvironment. In a cohort of 120 patients with advanced HNSCC, who were treated with primary or adjuvant radiotherapy, a significant association was found between relative p-AKT(Ser473) levels and overall survival (p = 0.006) as well as progression-free survival (p = 0.021), while no significant correlation was revealed for relative p-AKT(Thr308) levels. In ex vivo tissue cultures p-AKT(Ser473) levels were increased upon irradiation and treatment with the PI3K inhibitor LY294002 inhibited both basal and irradiation induced AKT(Ser473) phosphorylation. Strikingly, pretreatment with LY294002 sensitized tissue cultures derived from primary and recurrent tumors to radiotherapy as determined by impaired tumor cell proliferation and enhanced DNA damage. In conclusion, phosphorylation status of AKT(Ser473) in tumor specimens serves as a novel biomarker to identify patients with advanced HNSCC at high risk for treatment failure following radiotherapy, and our data from ex vivo tissue cultures support the assumption that pharmacological inhibition of AKT(Ser473) phosphorylation might circumvent radioresistance to improve efficiency and reduce toxicity of current treatment modalities.

Prognostic significance of spontaneous antibody responses against tumor-associated antigens in malignant melanoma patients.

Distribution, patterns and prognostic impact of spontaneous antibody responses against different tumor-associated antigens (TAAs) in malignant melanoma patients are unknown so far and were investigated in this study for the first time in a large cohort at different stages of the disease, identifying new prognostic biomarkers for malignant melanoma. Serum samples from 365 melanoma patients (97 Stage I melanoma patients, 87 Stage II, 92 Stage III and 89 Stage IV) and 100 age and gender matched healthy control donors were analyzed. Samples were drawn at the time of diagnosis (Stages I-III) or at time of diagnosis of distant metastasis (Stage IV). Applying a novel multiplex assay, humoral immune responses against 29 TAAs were determined and the association between response and patient survival was investigated. Antibody responses were mainly found in melanoma patients and all tested antigens elicited immune responses in all disease stages. Antibody responses against single antigens were either associated with poor prognosis and/or shorter progression-free survival (PFS) or had no influence. While in Stages I-III significant associations were observed between an antibody response and overall survival or PFS, among Stage IV patients, no significant association was found. Multivariate analyses identified specific humoral immune responses as prognostic factors independently of age, chemotherapy and immunotherapy. Antibody responses against specific TAA in Stage I-III melanoma patients correlate with poor prognosis and/or shorter PFS. These results may help to design clinical studies in order to evaluate the potential of these responses as prognostic serological biomarkers.

Molecular profiling of long-term survivors identifies a subgroup of glioblastoma characterized by chromosome 19/20 co-gain.

Glioblastoma (GBM) is a devastating tumor and few patients survive beyond 3 years. Defining the molecular determinants underlying long-term survival is essential for insights into tumor biology and biomarker identification. We therefore investigated homogeneously treated, IDH (wt) long-term (LTS, n = 10) and short-term survivors (STS, n = 6) by microarray transcription profiling. While there was no association of clinical parameters and molecular subtypes with long-term survival, STS tumors were characterized by differential polarization of infiltrating microglia with predominance of the M2 phenotype detectable both on the mRNA and protein level. Furthermore, transcriptional signatures of LTS and STS predicted patient outcome in a large, IDH (wt) cohort (n = 468). Interrogation of overlapping genomic alterations identified concurrent gain of chromosomes 19 and 20 as a favorable prognostic marker. The strong association of this co-gain with survival was validated by aCGH in a second, independent cohort (n = 124). Finally, FISH and gene expression data revealed gains to constitute low-amplitude, clonal events with a strong impact on transcription. In conclusion, these findings provide important insights into the manipulation of the innate immune system by particularly aggressive GBM tumors. Furthermore, we genomically characterize a previously unknown, clinically relevant subgroup of glioblastoma, which can easily be identified through modern neuropathological workup.

Pathogenic role of the eight probably/possibly carcinogenic HPV types 26, 53, 66, 67, 68, 70, 73 and 82 in cervical cancer.

Eight HPV types (HPV26, 53, 66, 67, 68, 70, 73 and 82) that are phylogenetically closely related to 12 WHO-defined high-risk (HR) HPV have been rarely but consistently identified as single HPV infections in about 3% of cervical cancer (CxCa) tissues. Due to lack of biological data, these types are referred to as probable/possible (p) HR-HPV. To analyse their biological activity in direct comparison to HR-HPV types, we selected 55 formalin-fixed, paraffin-embedded (FFPE) CxCa tissues harbouring single pHR-HPV infections (2-13 cases per type) and 266 tissues harbouring single HR-HPV (7-40 cases per type) from a worldwide, retrospective, cross-sectional study. Single HPV infection was verified by two genotyping methods. Presence of type-specific spliced E6*I mRNA transcripts and expression of cellular proteins indicative of HPV transformation were assessed in all cases. In 55 CxCa tissues with pHR-HPV, E6*I mRNA expression was 100%; high p16(INK4a) , 98%; low pRb, 96%; low CyD1, 93%; and low p53, 84%. Compared to HPV16 tissues as a reference, individual frequencies of these five markers did not differ significantly, either for any of the eight pHR-HPV and the 11 other HR types individually or for the groups of pHR and HR types without HPV16. We conclude that the eight pHR-HPV types, when present as a single infection in CxCa, are biologically active and affect the same cellular pathways as any of the fully recognized carcinogenic HR-HPV types. Therefore we have provided molecular evidence of carcinogenicity for types currently classified as probably/possibly carcinogenic. Although this evidence is crucial for HPV-type carcinogenicity classification, per se it is not sufficient for inclusion of these HPV types into population-wide primary and secondary prevention programmes. Such decisions have to include careful estimation of effectiveness and cost-benefit analyses.