Changes in gene expression patterns in the tumor microenvironment of head and neck squamous cell carcinoma under chemoradiotherapy depend on response

Chemoradiotherapy (CRT) is a standard treatment for advanced head and neck squamous cell carcinoma (HNSCC). Unfortunately, not all patients respond to this therapy and require further treatment, either salvage surgery or palliative therapy. The addition of immunotherapy to CRT is currently being investigated and early results describe a mixed response. Therefore, it is important to understand the impact of CRT on the tumor microenvironment (TME) to be able to interpret the results of the clinical trials. Paired biopsies from 30 HNSCC patients were collected before and three months after completion of primary CRT and interrogated for the expression of 1392 immune- and cancer-related genes. There was a relevant difference in the number of differentially expressed genes between the total cohort and patients with residual disease. Genes involved in T cell activation showed significantly reduced expression in these tumors after therapy. Furthermore, gene enrichment for several T cell subsets confirmed this observation. The analysis of tissue resident memory T cells (TRM) did not show a clear association with impaired response to therapy. CRT seems to lead to a loss of T cells in patients with incomplete response that needs to be reversed. It is not clear whether the addition of anti-PD-1 antibodies alone to CRT can prevent treatment failure, as no upregulation of the targets was measurable in the TME

Targeting the tumor mutanome for personalized vaccination in a TMB low non-small cell lung cancer.

BackgroundCancer is characterized by an accumulation of somatic mutations, of which a significant subset can generate cancer-specific neoepitopes that are recognized by autologous T cells. Such neoepitopes are emerging as important targets for cancer immunotherapy, including personalized cancer vaccination strategies.MethodsWe used whole-exome and RNA sequencing analysis to identify potential neoantigens for a patient with non-small cell lung cancer. Thereafter, we assessed the autologous T-cell reactivity to the candidate neoantigens using a long peptide approach in a cultured interferon gamma ELISpot and tracked the neoantigen-specific T-cells in the tumor by T-cell receptor (TCR) sequencing. In parallel, identified gene variants were incorporated into a Modified Vaccinia Ankara-based vaccine, which was evaluated in the human leucocyte antigen A*0201 transgenic mouse model (HHD).ResultsSequencing revealed a tumor with a low mutational burden: 2219 sequence variants were identified from the primary tumor, of which 23 were expressed in the transcriptome, involving 18 gene products. We could demonstrate spontaneous T-cell responses to 5/18 (28%) mutated gene variants, and further analysis of the TCR repertoire of neoantigen-specific CD4+ and CD8+ T cells revealed TCR clonotypes that were expanded in both blood and tumor tissue. Following vaccination of HHD mice, de novo T-cell responses were generated to 4/18 (22%) mutated gene variants; T cells reactive against two variants were also evident in the autologous setting. Subsequently, we determined the major histocompatibility complex restriction of the T-cell responses and used in silico prediction tools to determine the likely neoepitopes.ConclusionsOur study demonstrates the feasibility of efficiently identifying tumor-specific neoantigens that can be targeted by vaccination in tumors with a low mutational burden, promising successful clinical exploitation, with trials currently underway

Effects of epigenetic drugs on CD3+ CD8+ cytolytic cells of cancer patients

OBJECTIVE: This study aimed to investigate the effects of epigenetic drugs on antitumor peptide-specific CD8+ CTL clones isolated from patients with NSCLC and melanoma.MATERIALS AND METHODS: Three peptide-specific CD8+ CTL clones against hTERT and MAGE, isolated from patients with NSCLC and melanoma respectively were enrolled in this study. Four cancer cell lines, EBV transformed B cells and PBMCs were used as antigen presenting and feeder cells. The CTL clones were cultured in the presence of 5-aza-CdR and TSA and the alterations in the phenotypic, lytic, functional characteristics as well as in gene expression were investigated. Lytic capability was determined by standard chromium release assay and cell cycle kinetics was analysed using BrdU uptake. Apoptosis assay was performed using Annexin/7-AAD and the extracellular and intracellular protein expression was determined using flow cytometry. Gene expression was performed using quantitative RT-Real Time PCR.RESULTS: The epigenetic drugs had an immunomodulatory effect on antitumor peptide-specific CTL. It was confirmed that 5-aza-CdR decreased the global DNA methylation level of CTL by 47% and that the subtoxic concentrations did not affect the cell cycle. FAS cell surface expression was found to be increased by 47% whereas no significant change was observed in the expression of CD8, CD25, CD27, CD28, CD57 ,CD62L, CCR7, CD69, CD122, CD127 and CD178. CTL clones cultured in the presence of 5-aza-CdR exhibit a significant decrease in their cytolytic capabilities (up to70%) that is maintained after drug removal and a subsequent cycle of stimulation and culture (up to 90%). The molecular analysis of the clones against hTERT and MAGE determined that 5-aza-CdR increased gene expression with a fold change of 8 and 15.2 for FOXP3, 3.9 and 2.2 for FASL, 5.3 and 1.8 for FAS, 1.8 and 6.1 for TNF, 4.2 and 4.1 for PD-L1, 4.3 and 4.7 for PD-L2 respectively. No significant differences were observed for PERFORIN, GRANZYMEB, IFNG, IL1b, IL10, TNFSF10, TGFb1, TGFb3, TGFbR1, PD-1, SMAD2, SMAD3, SMAD4 and SMAD7. TSA was found to dramatically decrease the clone activation capability (up to 95.3%) in a dose-dependent manner as well as the clone lytic capability (up to 48.3%).CONCLUSION: The effects of epigenetic drugs on antitumor peptide-specific CD8+ CTL clones are multifaceted and it is difficult to produce definitive conclusions. Howbeit, the immunomodulatory effects observed with most prominent being a severely compromised lytic ability against targets presenting tumor peptides, lead to a phenotype with reduced ability to lyse target cancer cells.ΣΚΟΠΟΣ: Σκοπός της διατριβής ήταν η μελέτη της επίδρασης των επιγενετικών φαρμάκων σε πεπτιδο-ειδικούς CD8+ T-κυτταρολυτικούς κλώνους, ειδικούς για καρκινικά αντιγόνα, απομονωμένους από ασθενείς με καρκίνο του πνεύμονα και μελάνωμα. ΥΛΙΚΑ ΚΑΙ ΜΕΘΟΔΟΙ: Το υλικό της μελέτης αποτέλεσαν 3 πεπτιδο-ειδικοί CD8+ CTL κλωνοι με ειδικότητα έναντι πεπτιδίων της hTERT και του MAGE οι οποίοι απομονώθηκαν από ασθενείς με μη μικροκυτταρικό καρκίνο του πνεύμονα και μελάνωμα αντίστοιχα. Ως αντιγονοπαρουσιαστικά και κύτταρα τροφοί χρησιμοποιήθηκαν 4 καρκινικές σειρές, B-κύτταρα μετασχηματισμένα με EBV και PBMC. Τα επιγενετικά φάρμακα που χρησιμοποιήθηκαν ήταν η 5-aza-CdR και η TSA. Aναλύθηκε η επίδραση των φαρμάκων στη λυτική ικανότητα των CTL με τη δοκιμασία απελευθέρωσης 51Cr, στον κυτταρικό κύκλο με έλεγχο ενσωμάτωσης BrdU/7-ΑΑD, στην ικανότητα για απόπτωση με Annexin/7-AAD, στην επιφανειακή και ενδοκυττάρια έκφραση πρωτεϊνών με κυτταρομετρία ροής και στη γονιδιακή έκφραση με ποσοτική RT-Real Time PCR. ΑΠΟΤΕΛΕΣΜΑΤΑ: Τα επιγενετικά φάρμακα έχουν ανοσοδιαμορφωτική επίδραση στα αντικαρκινικά πεπτιδο-ειδικά CTL. Διαπιστώθηκε ότι η 5-aza-CdR μειώνει κατά 47% το ποσοστό της ολικής μεθυλίωσης του γενωμικού DNA και ότι οι δόσεις που χρησιμοποιήθηκαν δεν επηρεάζουν τον κυτταρικό κύκλο. Παρατηρήθηκε αύξηση κατά 47% στην επιφανειακή έκφραση FAS, ενώ δεν παρατηρήθηκε σημαντική διαφορά στην έκφραση των CD8, CD25, CD27, CD28, CD57 ,CD62L, CCR7, CD69, CD122, CD127 και CD178. Βρέθηκε σημαντική μείωση της κυτταρολυτικής ικανότητας (έως και 70%). Η μοριακή ανάλυση για τους κλώνους έναντι της hTERT και του MAGE αποκάλυψε ότι η 5-aza-CdR προκαλεί αύξηση της έκφρασης των: FOXP3 κατά 8x και 15.2x, του FASL κατά 3.9x και 2.2x, του FAS κατά 5.3x και 1.8x, του TNF κατά 1.8x και 6.1x, του PD-L1 κατά 4.2x και 4.1x και του PD-L2 κατά 4.3x και 4.7x αντίστοιχα. Δεν παρατηρήθηκαν σημαντικές διαφορές για τα μόρια: PERFORIN, GRANZYMEB, IFNG, IL1b, IL10, TNFSF10, TGFb1, TGFb3, TGFbR1, PD-1, SMAD2, SMAD3, SMAD4 και SMAD7. Η TSA φάνηκε να μειώνει δραματικά την ικανότητα ενεργοποίησης των κλώνων (έως και 95.3%) με δοσο-εξαρτώμενο τρόπο αλλά και την ικανότητα λύσης των κυττάρων στοχών (έως και 48.3%). ΣΥΜΠΕΡΑΣΜΑΤΑ: Οι αλλαγές που συντελούνται στα πεπτιδο-ειδικά έναντι του καρκίνου CTL από τα επιγενετικά φάρμακα είναι πολυδιάστατες και είναι δύσκολο να προτείνει κανείς οριστικά συμπεράσματα. Εντούτοις, φαίνεται ότι οι ανοσοδιαμορφωτικές επιδράσεις που παρατηρήσαμε και ειδικά η μείωση της ειδικής λυτικής ικανότητας των CTL οδηγούν σε ένα φαινότυπο με μειωμένη ικανότητα να λύει τα καρκινικά κύτταρα στόχους

Case of pediatric acquired chronic hepatocerebral degeneration

Acquired chronic hepatocerebral degeneration is a central nervous system disorder secondary to several conditions related to hepatic dysfunction. Clinical features of acquired chronic hepatocerebral degeneration include a hyperkinetic extrapyramidal syndrome, neuropsychiatric symptoms, or both. We present for the first time a pediatric case of acquired chronic hepatocerebral degeneration secondary to endstage biliary disease. The pediatric phenotype of acquired chronic hepatocerebral degeneration is presented, and the differential diagnosis in regard to Wilson's disease and management alternatives are discussed

Remote Rover Operations: Testing the Exomars Egress Case

This paper presents the results of the remote rover operations tests run on the 27-29th of October 2015 focused on the ExoMars egress manoeuvre scenario. A total of 5 differently challenging scenarios were tested in order to evaluate the capabilities of the operators with regards to the proper understanding of the criticality of each case that would allow them to make a sound decision on which egress direction to take. These experiments showed the usability of simulation tools 3DROCS&3DROV for acquiring the situational awareness needed for this purpose and the importance of planning and establishing the rules and conditions that enable the decision making process

Changes in Gene Expression Patterns in the Tumor Microenvironment of Head and Neck Squamous Cell Carcinoma Under Chemoradiotherapy Depend on Response

Chemoradiotherapy (CRT) is a standard treatment for advanced head and neck squamous cell carcinoma (HNSCC). Unfortunately, not all patients respond to this therapy and require further treatment, either salvage surgery or palliative therapy. The addition of immunotherapy to CRT is currently being investigated and early results describe a mixed response. Therefore, it is important to understand the impact of CRT on the tumor microenvironment (TME) to be able to interpret the results of the clinical trials. Paired biopsies from 30 HNSCC patients were collected before and three months after completion of primary CRT and interrogated for the expression of 1392 immune- and cancer-related genes. There was a relevant difference in the number of differentially expressed genes between the total cohort and patients with residual disease. Genes involved in T cell activation showed significantly reduced expression in these tumors after therapy. Furthermore, gene enrichment for several T cell subsets confirmed this observation. The analysis of tissue resident memory T cells (T(RM)) did not show a clear association with impaired response to therapy. CRT seems to lead to a loss of T cells in patients with incomplete response that needs to be reversed. It is not clear whether the addition of anti-PD-1 antibodies alone to CRT can prevent treatment failure, as no upregulation of the targets was measurable in the TME

Deleterious genetic variation across the NOD-signaling pathway is associated with reduced NFKB-signaling transcription and upregulation of alternative inflammatory transcripts in paediatric inflammatory bowel disease

Background: inflammatory bowel disease (IBD) may arise with inadequate immune response to intestinal bacteria. NOD2 is an established gene in Crohn’s disease pathogenesis, with deleterious variation associated with reduced NFKB-signaling. We hypothesised that deleterious variation across the NOD2-signaling pathway impacts on transcription.Methods: treatment-naïve paediatric IBD patients had ileal biopsies for targeted-autoimmune RNA-sequencing and blood for whole-exome-sequencing collected at diagnostic endoscopy. Utilising GenePy, a per individual, per gene score, genes within the NOD-signaling pathway were assigned a quantitative score representing total variant burden. Where multiple genes formed complexes, GenePy scores were summed to create a ‘complex’ score. Normalised transcript expression of 95-genes within this pathway were retrieved. Regression analysis was performed to determine the impact of genomic variation on gene transcription.Results: thirty-nine patients were included. Limited clustering of patients based on NOD-signaling transcripts was related to underlying genomic variation. Patients harbouring deleterious variation in NOD2 had reduced NOD2 (β=-0.702, p=4.3x10-5) and increased NFKBIA (β=0.486, p=0.001), reflecting reduced NFKB-signal activation. Deleterious variation in the NOD2-RIPK2 complex was associated with increased NLRP3 (β=0.8, p=3.1475x10-8) and TXN (β=-0.417, p=8.4x10-5) transcription, components of the NLRP3-inflammasome. Deleterious variation in the TAK1-TAB complex resulted in reduced MAPK14 transcription (β=-0.677, p=1.7x10-5), a key signal transduction protein in the NOD2-signaling cascade and increased IFNA1 (β=0.479, p=0.001), indicating reduced transcription of NFKB activators and alternative interferon transcription in these patients. Conclusions: data integration identified perturbation of NOD2-signaling transcription correlated with genomic variation. A hypoimmune NFKB-signaling transcription response was observed. Alternative inflammatory pathways were activated and may represent therapeutic targets in specific patients. <br/

Evaluating atezolizumab in patients with urinary tract squamous cell carcinoma (AURORA): study protocol for a single arm, open-label, multicentre, phase II clinical trial

Background: bladder and urinary tract cancers account for approximately 21,000 new diagnoses and 5,000 deaths annually in the UK. Approximately 90% are transitional cell carcinomas where advanced disease is treated with platinum based chemotherapy and PD-1/PD-L1 directed immunotherapy. Urinary tract squamous cell carcinoma (UTSCC) accounts for about 5% of urinary tract cancers overall making this a rare disease. We have yet to establish definitive systemic treatment options for advanced UTSCC. Preliminary translational data, from UTSCC patient tumour samples, indicate high PD-L1 expression and tumour infiltrating lymphocytes in a proportion of cases. Both of these features are associated with differential gene expression consistent with a tumour/immune microenvironment predicted to be susceptible to immune checkpoint directed immunotherapy which we will evaluate in the AURORA trial.Methods: AURORA is a single arm, open-label, multicentre,UK phase II clinical trial. 33 patients will be recruited from UK secondary care sites. Patients with UTSCC, suitable for treatment with palliative intent, will receive atezolizumab PD-L1 directed immunotherapy (IV infusion, 1680 mg, every 28 days) for one year if tolerated. Response assessment, by cross sectional imaging will occur every 12 weeks. AURORA uses a Simon's 2-stage optimal design with best overall objective response rate (ORR, by RECIST v1.1) at a minimum of 12 weeks from commencing treatment as the primary endpoint. Secondary endpoints will include overall survival, progression-free survival, duration of response, magnitude of response using waterfall plots of target lesion measurements, quality of life using the EORTC QLQ-C30 tool, safety and tolerability (CTCAE v5) and evaluation of potential biomarkers of treatment response including PD-L1 expression. Archival tumour samples and blood samples will be collected for translational analyses.Discussion: if this trial shows atezolizumab to be safe and effective it may lead to a future late phase randomised controlled trial in UTSCC. Ultimately, we hope to provide a new option for treatment for such patients.Trial registrations: EudraCT Number: 2021-001995-32 (issued 8th September 2021); ISRCTN83474167 (registered 11 May 2022); NCT05038657 (issued 9th September 2021).</p