IGF1R and TYRO3 as potential biomarkers for response prediction in malignant thymomas and thymic carcinomas treated with sunitinib

Metastatic and recurrent malignant thymomas (TH) and thymic carcinomas (TC) are generally refractory to current empiric radio chemotherapies, thus warranting the search for novel second-line therapeutic strategies. The multi-target tyrosine kinase inhibitor sunitinib has been shown to induce partial remissions and to prolong overall survival of patients with aggressive TH and TC. However, although sunitinib has been proposed as an alternative treatment option in refractory disease, treatment response is only partial, and not all patients benefit equally. For a more accurate response prediction, new biomarkers are needed. In this study, we used phospho receptor tyrosine kinase (RTK) and MAPK arrays as well as a multiplex tyrosine phosphorylation assay containing 144 kinase substrates to characterize malignant TH and TC and to generate a sunitinib response index (SRI) using sunitinib resistance-induced cell lines. The RTK and MAPK arrays stratified the patients into two main dominant phosphor patterns: Pattern 1 was characterized by a strong activation of EGFR while pattern 2 was typified by a strong activation of TYRO3/Dtk. This activation of TYRO3/Dtk correlated with recurrence and metastatic spread of malignant TH. p38 andRSK1 were cluster-specifically activated in the MAPKs but showed no specific disease correlation. The SRI was functionally validated in several cell lines, and the application of the SRI to native malignant TH and TC samples identified a potentially sunitinib-responsive and a potentially sunitinib-resistant group. Among the predicted upstream kinases, TYRO3/Dtk belonged to the top candidates responsible for sunitinib response. TYRO3/Dtk and the highly activated IGF-1R in responsive patients were also functionally validated. Specific siRNA knockdowns in sunitinib-resistant cell lines and overexpression confirmed the functional relevance of TYRO3/Dtk and IGF1-R for sunitinib resistance. We present the SRI as a new approach to the resistance prediction of sunitinib in malignant TH and TC and propose that the activity level of TYRO3/Dtk and IGF-1R could serve as markers to aid in treatment decisions. The results of this in vitro investigation need validation of the SRI in prospective clinical trials

Role of cancer stem cell markers ALDH1, BCL11B, BMI-1, and CD44 in the prognosis of advanced HNSCC

PURPOSE Cancer stem cells (CSCs) are held accountable for the progress of head and neck squamous cell carcinoma (HNSCC). In the presented study, the authors evaluated the prognostic value of CSC markers in two particular HNSCC cohorts. METHODS This two cohort study consisted of 85~patients with advanced stage HNSCC, treated with primary radio(chemo)therapy (pRCT), and 95~patients with HNSCC, treated with surgery and partially adjuvant radio(chemo)therapy. Overall survival (OS), disease-free survival (DFS), and disease-specific survival (DSS) were assessed. Samples were assessed for the expression of different molecular stem cell markers (ALDH1, BCL11B, BMI\hbox-1, and CD44). RESULTS In the pRCT cohort, none of the baseline patient and tumor features exhibited a~statistically significant relation with survival in either the cohort or the human papillomavirus (HPV)-stratified subcohorts. High expression of BMI\hbox-1 significantly decreased OS and DFS, while high expression of CD44 decreased all modes of survival. Multivariate analysis showed significant prognostic influence for all tested CSC markers, with high BMI\hbox-1 and CD44 decreasing survival (BMI-1: OS, DFS, DSS; CD44: OS, DFS) and high ALDH1 and BCL11B showing a~beneficial effect on survival (ALDH1: OS, DFS; BCL11B: OS, DSS). In the surgical cohort, classical prognosticators such as HPV status, R1 resection, and nodal status in HPV-negative HNSCC played a~significant role, but the tested CSC markers showed no significant effect on prognosis. CONCLUSION Although validation in independent cohorts is still needed, testing for CSC markers in patients with advanced or late stage HNSCC might be beneficial, especially if many comorbidities exist or disease is irresectable. The findings might guide the development and earlier use of targeted therapies in the future

Prognostic impact of additional HPV diagnostics in 102 patients with p16-stratified advanced oropharyngeal squamous cell carcinoma

Purpose!#!p16 overexpression was considered as surrogate marker to identify human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCCs).!##!Methods!#!102 patients with advanced stage OPSCCs treated primarily by transoral lasermicrosurgery were included. Prognostic associations of p16- and HPV-status were analyzed separately and combined.!##!Results!#!In contrast to p16, the HPV-status resulted in no significant survival discrepancies (5-year overall survival (OS) HPV-positive 64.9%, HPV-negative 78.7%). Combining both markers, p16-positive (p16-positive/HPV-positive, p16-positive/HPV-negative) and p16-negative/HPV-negative groups demonstrated comparable high survival (OS 78.1% vs. 85.6% vs. 73.6%). Lowest survival was observed for patients with p16-negative/HPV-positive OPSCCs (OS 40.8%). Never smoking patients with p16-positive OPSCCs demonstrated the highest survival, whereas within former/current smokers with p16-positive and p16-negative disease it was comparable low (OS 90.0% vs. 63.0% vs. 57.4%).!##!Conclusions!#!p16- and HPV-status should not be considered as equivalent markers for a better prognosis. Furthermore, they should not generally predominate patient associated factors like smoking

Atrial substrate characterization based on bipolar voltage electrograms acquired with multipolar, focal and mini-electrode catheters.

BACKGROUND Bipolar voltage (BV) electrograms for left atrial (LA) substrate characterization depend on catheter design and electrode configuration. AIMS The aim of the study was to investigate the relationship between the BV amplitude (BVA) using four catheters with different electrode design and to identify their specific LA cutoffs for scar and healthy tissue. METHODS AND RESULTS Consecutive high-resolution electroanatomic mapping was performed using a multipolar-minielectrode Orion catheter (Orion-map), a duo-decapolar circular mapping catheter (Lasso-map), and an irrigated focal ablation catheter with minielectrodes (Mifi-map). Virtual remapping using the Mifi-map was performed with a 4.5 mm tip-size electrode configuration (Nav-map). BVAs were compared in voxels of 3 × 3 × 3 mm3. The equivalent BVA cutoff for every catheter was calculated for established reference cutoff values of 0.1, 0.2, 0.5, 1.0, and 1.5 mV. We analyzed 25 patients (72% men, age 68 ± 15 years). For scar tissue, a 0.5 mV cutoff using the Nav corresponds to a lower cutoff of 0.35 mV for the Orion and of 0.48 mV for the Lasso. Accordingly, a 0.2 mV cutoff corresponds to a cutoff of 0.09 mV for the Orion and of 0.14 mV for the Lasso. For healthy tissue cutoff at 1.5 mV, a larger BVA cutoff for the small electrodes of the Orion and the Lasso was determined of 1.68 and 2.21 mV, respectively. CONCLUSION When measuring LA BVA, significant differences were seen between focal, multielectrode, and minielectrode catheters. Adapted cutoffs for scar and healthy tissue are required for different catheters

Validation of a clinical model for predicting left versus right ventricular outflow tract origin of idiopathic ventricular arrhythmias.

BACKGROUND Prediction of the chamber of origin in patients with outflow tract ventricular arrhythmias (OTVA) remains challenging. A clinical risk score based on age, sex and presence of hypertension was associated with a left ventricular outflow tract (LVOT) origin. We aimed to validate this clinical score to predict an LVOT origin in patients with OTVA. METHODS In a two-center observational cohort study, unselected patients undergoing catheter ablation (CA) for OTVA were enrolled. All procedures were performed using an electroanatomical mapping system. Successful ablation was defined as a ≥80% reduction of the initial overall PVC burden after 3 months of follow-up. Patients with unsuccessful ablation were excluded from this analysis. RESULTS We included 187 consecutive patients with successful CA of idiopathic OTVA. Mean age was 52 ± 15 years, 102 patients (55%) were female, and 74 (40%) suffered from hypertension. A LVOT origin was found in 64 patients (34%). A score incorporating age, sex and presence of hypertension reached 73% sensitivity and 67% specificity for a low (0-1) and high (2-3) score, to predict an LVOT origin. The combination of one ECG algorithm (V2 S/V3 R-index) with the clinical score resulted in a sensitivity and specificity of 81% and 70% for PVCs with R/S transition at V3 . CONCLUSION The published clinical score yielded a lower sensitivity and specificity in our cohort. However, for PVCs with R/S transition at V3, the combination with an existing ECG algorithm can improve the predictability of LVOT origin

Molecular Classification of Neuroendocrine Tumors of the Thymus

INTRODUCTION: The WHO classification of pulmonary neuroendocrine tumors (PNETs) is also used to classify thymic NETs (TNETs) into typical and atypical carcinoid (TC and AC), large cell neuroendocrine carcinoma (LCNEC), and small cell carcinoma (SCC), but little is known about the usability of alternative classification systems. METHODS: One hundred seven TNET (22 TC, 51 AC, 28 LCNEC, and 6 SCC) from 103 patients were classified according to the WHO, the European Neuroendocrine Tumor Society, and a grading-related PNET classification. Low coverage whole-genome sequencing and immunohistochemical studies were performed in 63 cases. A copy number instability (CNI) score was applied to compare tumors. Eleven LCNEC were further analyzed using targeted next-generation sequencing. Morphologic classifications were tested against molecular features. RESULTS: Whole-genome sequencing data fell into three clusters: CNIlow, CNIint, and CNIhigh. CNIlow and CNIint comprised not only TC and AC, but also six LCNECs. CNIhigh contained all SCC and nine LCNEC, but also three AC. No morphologic classification was able to predict the CNI cluster. Cases where primary tumors and metastases were available showed progression from low-grade to higher-grade histologies. Analysis of LCNEC revealed a subgroup of intermediate NET G3 tumors that differed from LCNEC by carcinoid morphology, expression of chromogranin, and negativity for enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). CONCLUSIONS: TNETs fall into three molecular subgroups that are not reflected by the current WHO classification. Given the large overlap between TC and AC on the one hand, and AC and LCNEC on the other, we propose a morphomolecular grading system, Thy-NET G1-G3, instead of histologic classification for patient stratification and prognostication. peerReviewe

Association between spatial distribution of leukocyte subsets and clinical presentation of head and neck squamous cell carcinoma

BackgroundInteractions between tumor cells and cells in the microenvironment contribute to tumor development and metastasis. The spatial arrangement of individual cells in relation to each other influences the likelihood of whether and how these cells interact with each other.MethodsThis study investigated the effect of spatial distribution on the function of leukocyte subsets in the microenvironment of human head and neck squamous cell carcinoma (HNSCC) using multiplex immunohistochemistry (IHC). Leukocyte subsets were further classified based on analysis of two previously published HNSCC single-cell RNA datasets and flow cytometry (FC).ResultsIHC revealed distinct distribution patterns of leukocytes differentiated by CD68 and CD163. While CD68hiCD163lo and CD68hiCD163hi cells accumulated near tumor sites, CD68loCD163hi cells were more evenly distributed in the tumor stroma. PD-L1hi and PD-1hi cells accumulated predominantly around tumor sites. High cell density of PD-L1hi CD68hiCD163hi cells or PD-1hi T cells near the tumor site correlated with improved survival. FC and single cell RNA revealed high variability within the CD68/CD163 subsets. CD68hiCD163lo and CD68hiCD163hi cells were predominantly macrophages (MΦ), whereas CD68loCD163hi cells appeared to be predominantly dendritic cells (DCs). Differentiation based on CD64, CD80, CD163, and CD206 revealed that TAM in HNSCC occupy a broad spectrum within the classical M1/M2 polarization. Notably, the MΦ subsets expressed predominantly CD206 and little CD80. The opposite was observed in the DC subsets.ConclusionThe distribution patterns and their distinct interactions via the PD-L1/PD-1 pathway suggest divergent roles of CD68/CD163 subsets in the HNSCC microenvironment. PD-L1/PD-1 interactions appear to occur primarily between specific cell types close to the tumor site. Whether PD-L1/PD-1 interactions have a positive or negative impact on patient survival appears to depend on both the spatial localization and the entity of the interacting cells. Co-expression of other markers, particularly CD80 and CD206, supports the hypothesis that CD68/CD163 IHC subsets have distinct functions. These results highlight the association between spatial leukocyte distribution patterns and the clinical presentation of HNSCC

Non-Mutational Key Features in the Biology of Thymomas

Thymomas (THs) are a unique group of heterogeneous tumors of the thymic epithelium. In particular, the subtypes B2 and B3 tend to be aggressive and metastatic. Radical tumor resection remains the only curative option for localized tumors, while more advanced THs require multimodal treatment. Deep sequencing analyses have failed to identify known oncogenic driver mutations in TH, with the notable exception of the GTF2I mutation, which occurs predominantly in type A and AB THs. However, there are multiple alternative non-mutational mechanisms (e.g., perturbed thymic developmental programs, metabolism, non-coding RNA networks) that control cellular behavior and tumorigenesis through the deregulation of critical molecular pathways. Here, we attempted to show how the results of studies investigating such alternative mechanisms could be integrated into a current model of TH biology. This model could be used to focus ongoing research and therapeutic strategies

Identification of Aeromonas veronii Genes Required for Colonization of the Medicinal Leech, Hirudo verbana▿

Most digestive tracts contain a complex consortium of beneficial microorganisms, making it challenging to tease apart the molecular interactions between symbiont and host. The digestive tract of Hirudo verbana, the medicinal leech, is an ideal model system because it harbors a simple microbial community in the crop, comprising the genetically amenable Aeromonas veronii and a Rikenella-like bacterium. Signature-tagged mutagenesis (STM) was used to identify genes required for digestive tract colonization. Of 3,850 transposon (Tn) mutants screened, 46 were identified as colonization mutants. Previously we determined that the complement system of the ingested blood remained active inside the crop and prevented serum-sensitive mutants from colonizing. The identification of 26 serum-sensitive mutants indicated a successful screen. The remaining 20 serum-resistant mutants are described in this study and revealed new insights into symbiont-host interactions. An in vivo competition assay compared the colonization levels of the mutants to that of a wild-type competitor. Attenuated colonization mutants were grouped into five classes: surface modification, regulatory, nutritional, host interaction, and unknown function. One STM mutant, JG736, with a Tn insertion in lpp, encoding Braun's lipoprotein, was characterized in detail. This mutant had a >25,000-fold colonization defect relative to colonization by the wild-type strain at 72 h and, in vitro, an increased sensitivity to sodium dodecyl sulfate, suggesting the presence of an additional antimicrobial property in the crop. The classes of genes identified in this study are consistent with findings from previous STM studies involving pathogenic bacteria, suggesting parallel molecular requirements for beneficial and pathogenic host colonization

Proteomic Comparison of Malignant Human Germ Cell Tumor Cell Lines

Malignant germ cell tumors (GCT) are the most common malignant tumors in young men between 18 and 40 years. The correct identification of histological subtypes, in difficult cases supported by immunohistochemistry, is essential for therapeutic management. Furthermore, biomarkers may help to understand pathophysiological processes in these tumor types. Two GCT cell lines, TCam-2 with seminoma-like characteristics, and NTERA-2, an embryonal carcinoma-like cell line, were compared by a quantitative proteomic approach using high-resolution mass spectrometry (MS) in combination with stable isotope labelling by amino acid in cell culture (SILAC). We were able to identify 4856 proteins and quantify the expression of 3936. 347 were significantly differentially expressed between the two cell lines. For further validation, CD81, CBX-3, PHF6, and ENSA were analyzed by western blot analysis. The results confirmed the MS results. Immunohistochemical analysis on 59 formalin-fixed and paraffin-embedded (FFPE) normal and GCT tissue samples (normal testis, GCNIS, seminomas, and embryonal carcinomas) of these proteins demonstrated the ability to distinguish different GCT subtypes, especially seminomas and embryonal carcinomas. In addition, siRNA-mediated knockdown of these proteins resulted in an antiproliferative effect in TCam-2, NTERA-2, and an additional embryonal carcinoma-like cell line, NCCIT. In summary, this study represents a proteomic resource for the discrimination of malignant germ cell tumor subtypes and the observed antiproliferative effect after knockdown of selected proteins paves the way for the identification of new potential drug targets