Transcriptome-Wide Gene Expression Profiles from FFPE Materials Based on a Nuclease Protection Assay Reveals Significantly Different Patterns between Synovial Sarcomas and Morphologic Mimickers.

BACKGROUND: Transcriptome profiling provides large data on tumor biology, which is particularly valuable in translational research and is becoming more and more important for clinical decision-making as well. RNA sequencing is considered to be the gold standard for this. However, FFPE material, as the most available material in routine pathology, has been an undefeatable obstacle for RNAseq. Extraction-free nuclease protection assays have the potential to be a reliable alternative method for large-scale expression profiling. The aim of this study was to validate and test the basic feasibility, technical applicability robustness, and reliability of the HTG transcriptome profiling (HTP) assay on clinical tumor samples. METHODS: FFPE samples from 44 synovial sarcomas (SyS) and 20 spindle cell sarcomas (SpcS) were used. The HTP assay was performed on 10 µm thin FFPE slides. After nuclease protection in the HTG Edge Seq System, libraries were generated for sequencing on an Illumina NextSeq 500 platform. Fastq data were parsed and then analyzed by using the HTG analysis platform EdgeSeq REVEAL. Immunohistochemistry was performed to validate the expression of TLE1. RESULTS: The technical application of the HTP Panel revealed robust and reliable results with 62 samples, and only 2 samples failed due to an incomplete digestion of gDNA. The analysis, performed at the analysis platform REVEAL, showed 5964 genes being significantly differentially expressed between SpcS and SyS. In particular, overexpression of the known marker TLE1 in synovial sarcoma could be recovered, which underlines the reliability of this system. DISCUSSION: Transcriptome profiling gets more and more important for tumor research and diagnostics. Among other established technologies, the HTP Panel has shown to be a feasible method to get robust and reliable results. Thereby, this method needs very few sample-input by getting a success-rate of 96.88%, which indicates the upper average range, compared to other technologies working with FFPE tissue. CONCLUSION: The nuclease protection assay-based HTP Panel is a feasible method for adequate transcriptome profiling with low sample input and therefore is suitable for further research of biomarkers.

During early stages of cancer, neutrophils initiate anti-tumor immune responses in tumor-draining lymph nodes.

Tumor-draining lymph nodes (LNs) play a crucial role during cancer spread and in initiation of anti-cancer adaptive immunity. Neutrophils form a substantial population of cells in LNs with poorly understood functions. Here, we demonstrate that, during head and neck cancer (HNC) progression, tumor-associated neutrophils transmigrate to LNs and shape anti-tumor responses in a stage-dependent manner. In metastasis-free stages (N0), neutrophils develop an antigen-presenting phenotype (HLA-DR+CD80+CD86+ICAM1+PD-L1-) and stimulate T cells (CD27+Ki67highPD-1-). LN metastases release GM-CSF and via STAT3 trigger development of PD-L1+ immunosuppressive neutrophils, which repress T cell responses. The accumulation of neutrophils in T cell-rich zones of LNs in N0 constitutes a positive predictor for 5-year survival, while increased numbers of neutrophils in LNs of N1-3 stages predict poor prognosis in HNC. These results suggest a dual role of neutrophils as essential regulators of anti-cancer immunity in LNs and argue for approaches fostering immunostimulatory activity of these cells during cancer therapy.

B-Helper Neutrophils in Regional Lymph Nodes Correlate with Improved Prognosis in Patients with Head and Neck Cancer.

The role of neutrophils during cancer formation and elimination is diverse. Here, for the first time, we investigate neutrophil helper cells (NBH), their influence on B cell activity in the regional lymph nodes (RLN) of head-and-neck cancer patients and the effect of this neutrophil/B cell interaction on patient prognosis. Circulating and RLN neutrophils of patients with stage I-IV head-and-neck squamous cell carcinoma were investigated with flow cytometry and qPCR. In addition, neutrophil/B cell co-localization in RLNs was evaluated using immunohistochemistry. B cell proliferation was assessed and correlated with the distance to neutrophils. Patient survival was evaluated. Neutrophils with the helper cell phenotype were identified in the RLN of HNC patients. B cells in close proximity to such NBH showed significantly higher proliferation rates, together with elevated activation-induced cytidine deaminase (AID) expression. Notably, patient survival was significantly higher in individuals with high NBH frequencies in the B follicles of RLNs. Neutrophils in RLN can support T cell-independent activation of the adaptive immune system through B cell stimulation, capturing helper cell phenotype character. The presence of such helper neutrophils in the RLNs of HNC patients positively correlates with patient prognosis.

Increased longevity due to sexual activity in mole-rats is associated with transcriptional changes in the HPA stress axis.

Sexual activity and/or reproduction are associated with a doubling of life expectancy in the long-lived rodent genus Fukomys. To investigate the molecular mechanisms underlying this phenomenon, we analyzed 636 RNA-seq samples across 15 tissues. This analysis suggests that changes in the regulation of the hypothalamic-pituitary-adrenal stress axis play a key role regarding the extended life expectancy of reproductive vs. non-reproductive mole-rats. This is substantiated by a corpus of independent evidence. In accordance with previous studies, the up-regulation of the proteasome and so-called 'anti-aging molecules', for example, dehydroepiandrosterone, is linked with enhanced lifespan. On the other hand, several of our results are not consistent with knowledge about aging of short-lived model organisms. For example, we found the up-regulation of the insulin-like growth factor 1/growth hormone axis and several other anabolic processes to be compatible with a considerable lifespan prolongation. These contradictions question the extent to which findings from short-lived species can be transferred to longer-lived ones.

Machine learning reveals a PD-L1-independent prediction of response to immunotherapy of non-small cell lung cancer by gene expression context.

OBJECTIVE: Current predictive biomarkers for PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1)-directed immunotherapy in non-small cell lung cancer (NSCLC) mostly focus on features of tumour cells. However, the tumour microenvironment and immune context are expected to play major roles in governing therapy response. Against this background, we set out to apply context-sensitive feature selection and machine learning approaches on expression profiles of immune-related genes in diagnostic biopsies of patients with stage IV NSCLC. METHODS: RNA expression levels were determined using the NanoString nCounter platform in formalin-fixed paraffin-embedded tumour biopsies obtained during the diagnostic workup of stage IV NSCLC from two thoracic oncology centres. A 770-gene panel covering immune-related genes and control genes was used. We applied supervised machine learning methods for feature selection and generation of predictive models. RESULTS: Feature selection and model creation were based on a training cohort of 55 patients with recurrent NSCLC treated with PD-1/PD-L1 antibody therapy. Resulting models identified patients with superior outcomes to immunotherapy, as validated in two subsequently recruited, separate patient cohorts (n = 67, hazard ratio = 0.46, p = 0.035). The predictive information obtained from these models was orthogonal to PD-L1 expression as per immunohistochemistry: Selecting by PD-L1 positivity at immunohistochemistry plus model prediction identified patients with highly favourable outcomes. Independence of PD-L1 positivity and model predictions were confirmed in multivariate analysis. Visualisation of the models revealed the predictive superiority of the entire 7-gene context over any single gene. CONCLUSION: Using context-sensitive assays and bioinformatics capturing the tumour immune context allows precise prediction of response to PD-1/PD-L1-directed immunotherapy in NSCLC.

The mismatch repair system is not affected in medullary thyroid carcinoma independent of stromal desmoplasia or ret proto-oncogene mutation.

INTRODUCTION: Medullary thyroid carcinoma (MTC) is an aggressive neuroendocrine neoplasia of the thyroid with 10 year overall survival of 50% and limited therapeutic options. High tumor mutational burden because of microsatellite instability (MSI) seems to be a predictor of response to immune checkpoint inhibitor therapy in different tumors. Therefor in 2017 the U.S. Food and Drug Administration (FDA) permitted the therapy of solid tumors with proven Microsatellite instability (MSI) with PD1 antibody Pembrolizumab independently of their origin. As little is known about MSI in MTC and new therapeutic strategies would be eligible we tried to find out, if therapy with PD1-inhibitors could be promising. MATERIAL AND METHODS: We performed MSI-analyses of 38 cases of MTC. Included were MTCs with and without stromal desmoplasia and with/without lymph node metastases. We also checked the immunhistochemical expression of PD-L-1 and performed next generation sequencing for genetic alterations. RESULTS: All cases revealed stable conditions of the microsatellites and showed immunohistochemically positive staining of the four mismatch repair proteins. PD-L-1- Immunostaining was negative in all cases. DISCUSSION: Our data show there is no MSI in MTCs, irrespectively of their status of desmoplasia, metastases and/or ret-mutation. Therefore a positive effect of PD1 inhibitors, because of MSI-associated high tumor mutational burden, seems to be unlikely.

MET Expression in Advanced Non-Small-Cell Lung Cancer: Effect on Clinical Outcomes of Chemotherapy, Targeted Therapy, and Immunotherapy.

BACKGROUND: The receptor tyrosine kinase MET is implicated in malignant transformation, tumor progression, metastasis, and acquired treatment resistance. We conducted an analysis of the effect of MET expression and MET genomic aberrations on the outcome of patients with advanced or metastatic pulmonary adenocarcinomas prospectively enrolled in an institutional precision oncology program. PATIENTS AND METHODS: Standardized immunohistochemistry (IHC) analyses of MET and markers of pathway activation were available in 384 patients, and next-generation sequencing-based MET hotspot mutation analyses were available from 892 patients. Clinical data were retrieved with a median follow-up from initial diagnosis of 37 months. RESULTS: High MET expression, defined as MET IHC 3+ or MET H-Score in the upper quartile, was observed in 102 of 384 patients (26.6%). MET exon 14 mutations were only detected in 7 of 892 patients (0.78%). High MET expression correlated with activation markers of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathways only in cases without Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR), v-Raf murine sarcoma viral oncogene homolog B (BRAF), anaplastic lymphoma kinase (ALK) and proto-oncogene tyrosine-protein kinase ROS (ROS1) aberrations. There was no association of MET expression with outcome during chemotherapy. High MET expression negatively affected the outcome during EGFR-targeting therapy but was associated with more favorable results with programmed death 1/programmed death ligand 1 (PD-L1)-directed therapy, independent of smoking history, PD-L1 expression or KRAS mutation. Two patients with MET exon 14 mutation and high PD-L1 expression failed to respond to pembrolizumab. CONCLUSION: MET expression affects the outcomes of targeted therapies in non-small-cell lung cancer, thus supporting the development of biomarker-informed combination strategies. The interaction of MET expression and MET mutation with immune checkpoint inhibitor therapy is novel and merits further investigation.

Phosphorylation of p70 Ribosomal Protein S6 Kinase ß-1 is an Independent Prognostic Parameter in Metastatic Colorectal Cancer.

BACKGROUND: Deregulation of signal transduction pathways plays a critical role in oncogenesis of colorectal cancer (CRC) and directly affects sensitivity to targeted therapies. Against this background we developed a comprehensive biomarker profiling program including markers of downstream signaling to study their association with clinical outcomes. PATIENTS AND METHODS: A prospectively studied cohort of 160 patients with metastatic CRC was included. Standard diagnostic workup included mutational analyses of Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral oncogene homolog (NRAS), and v-Raf murine sarcoma viral oncogene homolog B (BRAF). In addition, markers of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and mammalian target of rapamycin pathway activation (phosphorylation of extracellular signal-regulated kinase [ERK], AKT, and p70 ribosomal protein S6 kinase ß-1 [p70S6K]) were studied using standardized immunohistochemistry. RESULTS: There was a significant correlation between markers of ERK and AKT activation in the full cohort. In addition, phosphorylation of p70S6K correlated strongly with ERK and AKT phosphorylation and primary tumor localization in the right colon. Subgroup analyses specified these correlations to patients with all-RAS wild type tumors. In contrast, tumors harboring RAS mutations predominantly exhibited ERK phosphorylation. Interestingly, patients with CRC showing high p70S6K phosphorylation (highest quartile) had a significantly inferior overall survival (hazard ratio [HR], 2.4; P = .002) irrespective of RAS mutational status. This effect remained significant in multivariate analysis (P = .002). A patient subgroup characterized by high p70S6K phosphorylation and right-sided primary tumors had a particularly poor prognosis with a dramatically inferior overall survival (HR, 5.2; P < .001). Patients with right-sided primary tumor and low p70S6K phosphorylation had responses to anti-epidermal growth factor receptor antibody-based therapies and overall survival similar to patients with left-sided primary tumors. CONCLUSION: High phosphorylation of p70S6K is a novel, independent biomarker for poor prognosis, in particular in patients with right-sided primary tumors.

Molecular dissection of effector mechanisms of RAS-mediated resistance to anti-EGFR antibody therapy.

Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), cetuximab and panitumumab, are a mainstay of metastatic colorectal cancer (mCRC) treatment. However, a significant number of patients suffer from primary or acquired resistance. RAS mutations are negative predictors of clinical efficacy of anti-EGFR antibodies in patients with mCRC. Oncogenic RAS activates the MAPK and PI3K/AKT pathways, which are considered the main effectors of resistance. However, the relative impact of these pathways in RAS-mutant CRC is less defined. A better mechanistic understanding of RAS-mediated resistance may guide development of rational intervention strategies. To this end we developed cancer models for functional dissection of resistance to anti-EGFR therapy in vitro and in vivo. To selectively activate MAPK- or AKT-signaling we expressed conditionally activatable RAF-1 and AKT in cancer cells. We found that either pathway independently protected sensitive cancer models against anti-EGFR antibody treatment in vitro and in vivo. RAF-1- and AKT-mediated resistance was associated with increased expression of anti-apoptotic BCL-2 proteins. Biomarkers of MAPK and PI3K/AKT pathway activation correlated with inferior outcome in a cohort of mCRC patients receiving cetuximab-based therapy. Dual pharmacologic inhibition of PI3K and MEK successfully sensitized primary resistant CRC models to anti-EGFR therapy. In conclusion, combined targeting of MAPK and PI3K/AKT signaling, but not single pathways, may be required to enhance the efficacy of anti-EGFR antibody therapy in patients with RAS-mutated CRC as well as in RAS wild type tumors with clinical resistance.