Immunhistochemische Untersuchungen zur Expression von Tumormarkern und Wachstumsfaktorrezeptoren bei Hunden mit malignen Nasentumoren

Einleitung: Nasenhöhlentumoren stellen mit bis zu 47 % die Hauptursache für Nasenausfluss beim Hund dar. Sie sind überwiegend maligne, zu 60 % Karzinome und zu 34 % Sarkome. Die mediane Überlebenszeit (MÜZ) liegt ohne Therapie bei etwa drei Monaten. Nach einer Bestrahlungstherapie beträgt sie etwa 8-20 Monate. Eine Therapie mit klassischen Chemotherapeutika oder eine Tumorablation über eine offen-chirurgische Rhinotomie führen nicht zu einer Verlängerung der Überlebenszeit. Durch ein neues Therapieverfahren, die endoskopisch interventionelle Zytoreduktion (EIZ), werden bei deutlich weniger Nebenwirkungen und Sitzungen in Allgemeinanästhesie ähnliche Überlebenszeiten erreicht wie durch eine Radiotherapie. Da es sich bei der EIZ um eine Zytoreduktion handelt, bei der die Nasenhöhlentumoren nicht mit Sicherheitsabstand im gesunden Gewebe entfernt werden können, stellt sich die Frage, ob die Überlebenszeit zusätzlich durch adjuvante Therapeutika verlängert werden kann. Als solche kommen beispielsweise Tyrosinkinase-Inhibitoren (TKI) und Cyclooxygenase-2 (COX-2)-Inhibitoren infrage, deren Zielstruktur-Expression besonders in kaninen nasalen Sarkomen noch unbekannt ist. Ziele der Untersuchungen: Ziel dieser Arbeit ist, anhand von Bioptaten von kaninen nasalen Karzinomen und Sarkomen eine immunhistochemische Charakterisierung durchzuführen. Hierzu wurden 10 Marker ausgewählt. Besonders im Fokus standen die Wachstumsfaktorrezeptoren vascular endothelial growth factor receptor-2 (VEGFR-2) und epidermal growth factor receptor (EGFR) sowie COX-2, die ersten beiden als Zielstrukturen von TKI und der letztere als Zielstruktur von COX-2-Inhibitoren. So soll eine Wirksamkeit dieser Medikamente bei kaninen nasalen Karzinomen und Sarkomen evaluiert werden. Weiterhin soll eine Korrelation zwischen der Expression von bestimmten Markern (p53, Ki-67, aktivierte Caspase-3, Survivin, E-Cadherin) mit den klinischen Daten zur Tumorkategorie und Überlebenszeit der Patienten untersucht werden. Außerdem soll als Grundlage für den Einsatz neuartiger Medikamente analysiert werden, ob EGFR, VEGFR-2 oder COX-2 in Karzinomen und Sarkomen unterschiedlich stark exprimiert werden. Tiere, Material und Methoden: Es wurden 19 Karzinome, sieben Sarkome und drei andere Tumorarten (ein malignes Melanom, zwei undifferenzierte maligne Tumoren unklarer Histogenese) retrospektiv immunhistochemisch auf die Expression von EGFR, VEGFR-2, COX-2, p53, Ki-67, aktivierter Caspase-3, Survivin, E-Cadherin, Zytokeratinen und Vimentin untersucht. Von drei Patienten wurden insgesamt vier Rezidivbioptate entnommen und ebenfalls immunhistochemisch untersucht. Beidseitige Nasenschleim-hautbioptate von neun gesunden Beagles dienten als Kontrollgruppe (genehmigungspflichtiger Tierversuch: TVV 02/18, Landesdirektion Sachsen). Alle Bioptate wurden während einer standardisierten Diagnostik mit computer-/ magnetresonanztomographischer Untersuchung - bei der auch ein Staging in vier Tumor-Kategorien (T1-T4) durchgeführt wurde - und Rhinoskopie zwischen Jan. 2015 und Dez. 2018 entnommen. Die immunhistochemische Untersuchung der in Formalin fixierten und in Paraffin eingebetteten Gewebeschnitte wurde mit der Avidin-Biotin-Komplex-Methode durchgeführt, nachdem die histopathologischen Diagnosen an Hämatoxylin-Eosin-gefärbten Schnitten gestellt wurden. Die immun-histochemischen Färbungen wurden entweder quantitativ oder semiquantitativ ausgewertet. Die Ergebnisse wurden auf Normalverteilung getestet und u.a. mit One-way Anova oder Kruskal-Wallis Test analysiert. Die MÜZ wurde mit der Kaplan Meier Methode berechnet und mit Log-Rank Test und Gehan-Breslow-Wilcoxon Test verglichen (Signifikanzniveau alpha = 5 %). Ergebnisse: 29 Hunde haben die Einschlusskriterien erfüllt. 14 Hunde wurden unmittelbar nach der Diagnostik euthanasiert; 15 Hunde wurden mit einer EIZ behandelt. Die MÜZ der Patienten in T1 (n = 3) nach EIZ betrug 1362 Tage und war signifikant länger als die MÜZ der Patienten in T2 (n = 1) mit 379 Tagen, in T3 (n = 8) mit 250 Tagen und in T4 (n = 1) mit 75 Tagen (p = 0,0062). Von den nasalen Karzinomen zeigten 68 % für EGFR, 100 % für VEGFR-2, 63 % für COX-2, 100 % für Survivin und 100 % für E-Cadherin eine immunhistochemisch positive Reaktion. Von den nasalen Sarkomen reagierten 100 % für VEGFR-2, 57 % für COX-2 und 86 % für Survivin positiv. Die Proteine EGFR und E-Cadherin werden ausschließlich von epithelialen Zellen exprimiert. Die Expression lag somit bei den vorliegenden Sarkomen bei 0 %. Unter den anderen Tumoren waren 33 % für EGFR, 100 % für VEGFR-2, 67 % für COX-2, 67 % für Survivin und 67 % für E-Cadherin positiv. Die mediane Expression von p53 lag bei 0,9 %, von Ki-67 bei 25 % und von aktivierter Caspase-3 bei 0,7 %. Die Unterschiede in der Expression von EGFR, VEGFR-2, COX-2, p53, Ki-67, aktivierter Caspase-3, Survivin und E-Cadherin zwischen den einzelnen histogenetischen Gruppen sowie zwischen den vier Tumor-Kategorien und in der MÜZ waren nicht signifikant. Eine Korrelation der VEGFR-2-Expression mit der MÜZ oder den T-Kategorien konnte nicht untersucht werden, da alle Tumoren der drei histogenetischen Gruppen VEGFR-2-positiv waren. 100 % der Karzinome zeigten eine Zytokeratin-Expression, 0 % eine Vimentin-Expression. Sarkome verhielten sich dazu konträr. In den anderen Tumoren konnten weder Zytokeratine noch Vimentin immunhistochemisch nachgewiesen werden. In den Rezidivbioptaten war ein Anstieg der COX-2 und aktivierte-Caspase-3-Expression zu beobachten, der aufgrund der geringen Fallzahl nicht statistisch untersucht werden konnte. Schlussfolgerungen: In der vorliegenden Studie wurden erstmalig kanine nasale Karzinome und Sarkome vergleichend immunhistochemisch untersucht. Weiterhin konnte erstmalig gezeigt werden, dass auch mesenchymale und andere Tumoren in vergleichbarer Häufigkeit wie Karzinome der Nase COX-2 exprimieren, wodurch ein Einsatz von COX-2-Inhibitoren nach einer Zytoreduktion bei Nasenhöhlentumoren allgemein von Nutzen sein könnte. Da alle Tumoren VEGFR-2 und die Mehrzahl der Karzinome (68 %) EGFR exprimierten, könnte eine adjuvante Therapie nach EIZ durch einen TKI mit VEGFR-2 oder EGFR als Zielstruktur einen positiven Einfluss auf die Überlebenszeit der erkrankten Hunde haben. Durch die Expression von E-Cadherin und Zytokeratinen in 100 % der Karzinome und 0 % der Sarkome sowie der Expression von Vimentin in 0 % der Karzinome und 100 % der Sarkome konnte die histopathologische Diagnose im Hinblick auf die Histogenese der Tumoren bestätigt werden. Auf der Grundlage der Ergebnisse der vorliegenden Studie sollte eine klinische Studie zur Anwendung von TKI und COX-2-Inhibitoren zur Untersuchung der klinischen Wirksamkeit und Sicherheit bei nasalen Tumoren von Hunden durchgeführt werden.:1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 Physiologie der Nasenhöhle 2 2.1.1 Anatomischer und histologischer Aufbau 2 2.1.2 Funktionen der Nasenhöhle und Nasenschleimhaut 3 2.2 Tumoren der Nase und der Nasennebenhöhlen 3 2.2.1 Prävalenz und Signalement von Hunden mit Nasentumoren 3 2.2.2 Biologisches Verhalten der Tumoren 3 2.3 Klinische Symptome 4 2.4 Diagnostik 5 2.4.1 Laboruntersuchungen 5 2.4.2 Bildgebende Verfahren 6 2.4.3 Rhinoskopie 9 2.4.4 Histopathologische Untersuchung 10 2.5 Therapieoptionen 10 2.5.1 Radiotherapie 10 2.5.2 Rhinotomie 11 2.5.3 Chemotherapie 11 2.5.4 Endoskopisch interventionelle Zytoreduktion (EIZ) 12 2.5.5 Tyrosinkinase-Inhibitoren 13 2.5.6 Antikörper gegen Rezeptortyrosinkinasen 15 2.5.7 Cyclooxigenase-Inhibitoren 16 2.6 Prognose 16 2.7 Zielantigene für die Immunhistochemie 17 2.7.1 Epidermal growth factor receptor (EGFR) 17 2.7.2 Vascular endothelial growth factor receptor-2 (VEGFR-2) 18 2.7.3 Cyclooxygenase-2 (COX-2) 18 2.7.4 p53 19 2.7.5 Ki-67 19 2.7.6 Aktivierte Caspase-3 20 2.7.7 Survivin 20 2.7.8 E-Cadherin 21 2.7.9 Zytokeratine 21 2.7.10 Vimentin 21 3 HUNDE, MATERIAL UND METHODEN 22 3.1 Patienten 22 3.2 Bioptate und Einschlusskriterien 23 3.3 Kontrolltiere 24 3.4 Immunhistochemische Untersuchungen 25 3.5 Auswertung der immunhistochemischen Reaktionen 28 3.6 Statistische Auswertung 29 4 ERGEBNISSE 31 4.1 Patienten 31 4.1.1 Signalement und Anamnese 31 4.1.2 Einteilung der Patienten in T-Kategorien 33 4.1.3 Histopathologische Befunde 34 4.1.4 Mediane Überlebenszeit nach endoskopisch interventioneller Zytoreduktion 34 4.1.5 Gesunde Kontrollgruppe 35 4.2 Ergebnisse der immunhistochemischen Untersuchungen 37 4.2.1 Kontrollen und Absorptionsreaktionen 37 4.2.2 Epidermal growth factor receptor (EGFR) 37 4.2.3 Vascular endothelial growth factor receptor-2 (VEGFR-2) 40 4.2.4 Cyclooxygenase-2 (COX-2) 42 4.2.5 p53 46 4.2.6 Ki-67 48 4.2.7 Aktivierte Caspase-3 50 4.2.8 Survivin 52 4.2.9 E-Cadherin 55 4.2.10 Zytokeratine 58 4.2.11 Vimentin 58 4.2.12 Bioptate von Tumorrezidiven 60 5 DISKUSSION 62 6 ZUSAMMENFASSUNG 84 7 SUMMARY 86 8 LITERATURVERZEICHNIS 88 9 ANHANG 105 9.1 Übersicht über die Hunde und Bioptate 105 9.2 Ergebnistabellen 107 9.3 Immunhistochemisches Reaktionsprotokoll 120 9.4 Ansatz der Lösungen und Puffer für die Immunhistochemie 122 9.5 Bezugsquellen für Geräte, Einmalartikel, Reagenzien und Chemikalien 123 9.6 Abbildungs- und Tabellenverzeichnis 125Introduction: Tumours of the nasal cavity are the main cause of nasal discharge in dogs (up to 47 %). They are almost always malignant, 60 % are carcinomas and 34 % are sarcomas. Without performing any treatment, the median survival time (MST) is about three months. After radiation therapy, the MST is about 8-20 months. A therapy with conventional chemotherapeutics or tumour ablation via open surgical rhinotomy does not prolong the survival time. A new treatment method, the endoscopic interventional cytoreduction (EIC), achieves survival times similar to radiotherapy with considerably fewer sessions under general anaesthesia and fewer potential adverse events. As EIC is a cytoreduction procedure in which the intranasal tumours cannot be removed with safety margins in surrounding healthy tissue, the question arises whether survival could be prolonged by an additional application of adjuvant therapeutics. As such, for example, tyrosine kinase inhibitors (TKIs) and cyclooxygenase-2 (COX-2) inhibitors may be considered, whose target expression is still unknown, especially in canine nasal sarcomas. Aims of the study: One aim of this study is to perform an immunohistochemical characterisation on the basis of biopsy specimens from canine nasal carcinomas and sarcomas. For this purpose, 10 markers were selected. We particularly focused on the growth factor receptors vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) as well as COX-2, the first two being targets of TKIs and the latter one being the target of COX-2-inhibitors. Thus, a possible efficacy of these drugs in canine nasal carcinomas and sarcomas should be evaluated. Furthermore, a correlation between the expression of specific markers (p53, Ki-67, cleaved caspase-3, survivin, E-cadherin) with clinical data of the tumour stage and patient survival time should be investigated. Moreover, it will be analysed as a basis for the use of novel drugs whether EGFR, VEGFR-2 or COX-2 are differentially expressed in carcinomas and sarcomas. Animals, Material and Methods: A total of 19 carcinomas, seven sarcomas and three other tumour types (one malignant melanoma, two undifferentiated malignant tumorus of unclear histogenesis) were retrospectively examined by immunohistochemistry for the expression of EGFR, VEGFR-2, COX-2, p53, Ki-67, cleaved caspase-3, survivin, E-cadherin, cytokeratins and vimentin. A total of four biopsies from recurrent tumours were obtained from three patients and also examined by immunohistochemistry. Bilateral nasal mucosal samples from nine healthy beagles served as a control group (animal experiment requiring approval: TVV 02/18, Directorate of the Federal State of Saxony, Germany). All biopsy specimens were collected during a standardised diagnostic procedure with computer /magnetic resonance tomography, which also included a staging into four tumour stages (T1-T4) and rhinoscopy between Jan 2015 and Dec 2018. The immunohistochemical examination of tissue sections fixed in formalin and embedded in paraffin was performed using the avidin-biotin complex method after histopathological diagnoses had been made on haematoxylin-eosin stained sections. The immunohistochemically stained sections were evaluated either quantitatively or semiquantitatively. Results were tested for normal distribution and analysed by the one-way anova or Kruskal-Wallis test, among others. MST was calculated using the Kaplan Meier method and compared with the log-rank test and Gehan-Breslow-Wilcoxon test (significance level alpha = 5 %). Results: A total of 29 dogs met the inclusion criteria. A total of 14 dogs were euthanised immediately after diagnosis; 15 dogs were treated with an EIC. The MST of patients in T1 (n = 3) after EIC was 1362 days and was significantly longer than the MST of those patients in T2 (n = 1) at 379 days, T3 (n = 8) at 250 days and T4 (n = 1) at 75 days (p = 0.0062). Of the nasal carcinomas, 68 % were immunohistochemically positive for EGFR, 100 % for VEGFR-2, 63 % for COX-2, 100 % for survivin and 100 % for E-cadherin. Of the nasal sarcomas, 100 % reacted positively for VEGFR-2, 57 % for COX-2 and 86 % for survivin. The proteins EGFR and E-cadherin were expressed exclusively by epithelial cells and, therefore, the expression was 0 % in the present sarcomas. Amongst the other tumours, 33 % were positive for EGFR, 100 % for VEGFR-2, 67 % for COX-2, 67 % for survivin and 67 % for E-cadherin. The median expression of p53 was 0.9 %, that of Ki-67 25 % and that of cleaved caspase-3 0.7 %. Differences in expression of EGFR, VEGFR-2, COX-2, p53, Ki-67, cleaved caspase-3, survivin and E-cadherin among the histogenetic groups, the four tumour stages and in MST were not significant. However, a correlation of VEGFR-2 expression with MST or the tumour stages could not be investigated because all tumours in the three histogenetic groups were VEGFR-2 positive. A total of 100 % of carcinomas showed cytokeratin expression, and 0 % showed vimentin expression. Sarcomas behaved in a contrary manner. In the other tumours, neither cytokeratins nor vimentin could be detected by immunohistochemistry. An increase in COX-2 and cleaved caspase-3 expression was observed in the recurrent tumour biopsies, which could not be statistically investigated due to the small number of cases. Conclusions: In the present study, canine nasal carcinomas and sarcomas were investigated comparatively by immunohistochemistry for the first time. Again, it was shown for the first time that mesenchymal and other tumours also express COX-2 at comparable frequencies to carcinomas of the nose, suggesting that the use of COX-2 inhibitors after cytoreduction may be of general benefit in nasal cavity tumours. As all tumours expressed VEGFR-2 and the majority of carcinomas (68 %) expressed EGFR, the adjuvant therapy after EIC by a TKI targeting VEGFR-2 or EGFR could have a beneficial effect on the survival of diseased dogs. The expression of E-cadherin and cytokeratins in 100 % of the carcinomas and 0 % of the sarcomas as well as the expression of vimentin in 0 % of the carcinomas and 100 % of the sarcomas confirmed the histopathological diagnosis with regard to the histogenesis of the tumours. Based on these results of the present study, a clinical trial should be performed on the use of TKIs and COX-2 inhibitors to investigate the clinical efficacy and safety of canine nasal tumours.:1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 Physiologie der Nasenhöhle 2 2.1.1 Anatomischer und histologischer Aufbau 2 2.1.2 Funktionen der Nasenhöhle und Nasenschleimhaut 3 2.2 Tumoren der Nase und der Nasennebenhöhlen 3 2.2.1 Prävalenz und Signalement von Hunden mit Nasentumoren 3 2.2.2 Biologisches Verhalten der Tumoren 3 2.3 Klinische Symptome 4 2.4 Diagnostik 5 2.4.1 Laboruntersuchungen 5 2.4.2 Bildgebende Verfahren 6 2.4.3 Rhinoskopie 9 2.4.4 Histopathologische Untersuchung 10 2.5 Therapieoptionen 10 2.5.1 Radiotherapie 10 2.5.2 Rhinotomie 11 2.5.3 Chemotherapie 11 2.5.4 Endoskopisch interventionelle Zytoreduktion (EIZ) 12 2.5.5 Tyrosinkinase-Inhibitoren 13 2.5.6 Antikörper gegen Rezeptortyrosinkinasen 15 2.5.7 Cyclooxigenase-Inhibitoren 16 2.6 Prognose 16 2.7 Zielantigene für die Immunhistochemie 17 2.7.1 Epidermal growth factor receptor (EGFR) 17 2.7.2 Vascular endothelial growth factor receptor-2 (VEGFR-2) 18 2.7.3 Cyclooxygenase-2 (COX-2) 18 2.7.4 p53 19 2.7.5 Ki-67 19 2.7.6 Aktivierte Caspase-3 20 2.7.7 Survivin 20 2.7.8 E-Cadherin 21 2.7.9 Zytokeratine 21 2.7.10 Vimentin 21 3 HUNDE, MATERIAL UND METHODEN 22 3.1 Patienten 22 3.2 Bioptate und Einschlusskriterien 23 3.3 Kontrolltiere 24 3.4 Immunhistochemische Untersuchungen 25 3.5 Auswertung der immunhistochemischen Reaktionen 28 3.6 Statistische Auswertung 29 4 ERGEBNISSE 31 4.1 Patienten 31 4.1.1 Signalement und Anamnese 31 4.1.2 Einteilung der Patienten in T-Kategorien 33 4.1.3 Histopathologische Befunde 34 4.1.4 Mediane Überlebenszeit nach endoskopisch interventioneller Zytoreduktion 34 4.1.5 Gesunde Kontrollgruppe 35 4.2 Ergebnisse der immunhistochemischen Untersuchungen 37 4.2.1 Kontrollen und Absorptionsreaktionen 37 4.2.2 Epidermal growth factor receptor (EGFR) 37 4.2.3 Vascular endothelial growth factor receptor-2 (VEGFR-2) 40 4.2.4 Cyclooxygenase-2 (COX-2) 42 4.2.5 p53 46 4.2.6 Ki-67 48 4.2.7 Aktivierte Caspase-3 50 4.2.8 Survivin 52 4.2.9 E-Cadherin 55 4.2.10 Zytokeratine 58 4.2.11 Vimentin 58 4.2.12 Bioptate von Tumorrezidiven 60 5 DISKUSSION 62 6 ZUSAMMENFASSUNG 84 7 SUMMARY 86 8 LITERATURVERZEICHNIS 88 9 ANHANG 105 9.1 Übersicht über die Hunde und Bioptate 105 9.2 Ergebnistabellen 107 9.3 Immunhistochemisches Reaktionsprotokoll 120 9.4 Ansatz der Lösungen und Puffer für die Immunhistochemie 122 9.5 Bezugsquellen für Geräte, Einmalartikel, Reagenzien und Chemikalien 123 9.6 Abbildungs- und Tabellenverzeichnis 12

Emerging role of circulating tumor cells in immunotherapy.

Over the last few years, immunotherapy, in particular, immune checkpoint inhibitor therapy, has revolutionized the treatment of several types of cancer. At the same time, the uptake in clinical oncology has been slow owing to the high cost of treatment, associated toxicity profiles and variability of the response to treatment between patients. In response, personalized approaches based on predictive biomarkers have emerged as new tools for patient stratification to achieve effective immunotherapy. Recently, the enumeration and molecular analysis of circulating tumor cells (CTCs) have been highlighted as prognostic biomarkers for the management of cancer patients during chemotherapy and for targeted therapy in a personalized manner. The expression of immune checkpoints on CTCs has been reported in a number of solid tumor types and has provided new insight into cancer immunotherapy management. In this review, we discuss recent advances in the identification of immune checkpoints using CTCs and shed light on the potential applications of CTCs towards the identification of predictive biomarkers for immunotherapy

Does multidisciplinary videoconferencing between a head-and-neck cancer centre and its partner hospital add value to their patient care and decision-making? A mixed-method evaluation

OBJECTIVES: Given the difficulties in diagnosing and treating head-and-neck cancer, care is centralised in the Netherlands in eight head-and-neck cancer centres and six satellite regional hospitals as preferred partners. A requirement is that all patients of the partner should be discussed in a multidisciplinary team meeting (MDT) with the head-and-neck centre as part of a Dutch health policy rule. In this mixed-method study, we evaluate the value that the video-conferenced MDT adds to the MDTs in the care pathway, quantitative regarding recommendations given and qualitative in terms of benefits for the teams and the patient. DESIGN: A sequential mixed-method study. SETTING: One oncology centre and its partner in the Northern part of the Netherlands. PARTICIPANTS: Head-and-neck cancer specialists presenting patient cases during video-conferenced MDT over a period of 6 months. Semistructured interviews held with six medical specialists, three from the centre and three from the partner. PRIMARY AND SECONDARY OUTCOME MEASURES: Percentage of cases in which recommendations were given on diagnostic and/or therapeutic plans during video-conferenced MDT. RESULTS: In eight of the 336 patient cases presented (2%), specialists offered recommendations to the collaborating team (three given from centre to partner and five from partner to centre). Recommendations mainly consisted of alternative diagnostic modalities or treatment plans for a specific patient. Interviews revealed that specialists perceive added value in discussing complex cases because the other team offered a fre

Medical-Data-Models.org:A collection of freely available forms (September 2016)

MDM-Portal (Medical Data-Models) is a meta-data repository for creating, analysing, sharing and reusing medical forms, developed by the Institute of Medical Informatics, University of Muenster in Germany. Electronic forms for documentation of patient data are an integral part within the workflow of physicians. A huge amount of data is collected either through routine documentation forms (EHRs) for electronic health records or as case report forms (CRFs) for clinical trials. This raises major scientific challenges for health care, since different health information systems are not necessarily compatible with each other and thus information exchange of structured data is hampered. Software vendors provide a variety of individual documentation forms according to their standard contracts, which function as isolated applications. Furthermore, free availability of those forms is rarely the case. Currently less than 5 % of medical forms are freely accessible. Based on this lack of transparency harmonization of data models in health care is extremely cumbersome, thus work and know-how of completed clinical trials and routine documentation in hospitals are hard to be re-used. The MDM-Portal serves as an infrastructure for academic (non-commercial) medical research to contribute a solution to this problem. It already contains more than 4,000 system-independent forms (CDISC ODM Format, www.cdisc.org, Operational Data Model) with more than 380,000 dataelements. This enables researchers to view, discuss, download and export forms in most common technical formats such as PDF, CSV, Excel, SQL, SPSS, R, etc. A growing user community will lead to a growing database of medical forms. In this matter, we would like to encourage all medical researchers to register and add forms and discuss existing forms

Utility of a liquid biopsy to identify intra-tumoural molecular heterogeneity in head and neck squamous cell carcinoma: the role of circulating tumour cells

BACKGROUND: Circulating tumour cells (CTCs) in the blood of cancer patients are a potentially rich source of biomarkers to guide cancer therapy, particularly when tumours cannot be sampled directly. The optimal method of CTC enrichment/isolation and subsequent downstream characterisation remains unclear. Current marker dependent platforms are biased towards specific CTC sub-groups, and CTC characterisation focuses on genomic characterisation, being unable to detect post-transcriptional modification. AIMS: The aims of this project were: i) develop blood sampling methods able to preserve key CTC parameters with minimal sampling handling, ii) optimise a marker-independent CTC enrichment technique, the Parsortix microfluidic platform, for use in head and neck squamous cell carcinoma (HNSCC), iii) investigate methods for direct proteomic characterisation of CTCs to detect relevant post translational modifications. Collectively these aims would streamline sample collection thus simplifying and enhancing national/international multi-centre CTC research studies and trials while allowing deeper characterisation of CTC biology. METHODS: A mock CTC model using the FaDu and SCC047 HNSCC cell lines spiked into healthy donor blood was developed and used to optimise the Parsortix platform and downstream immunofluorescence microscopy and flow cytometry characterisation. Subsequently, a mass cytometry antibody panel of 44 markers (including epithelial/EMT, proliferative, stemness, immune and phosphorylated signalling proteins) was optimised for staining quality on Transfix fixed cells, following Parsortix enrichment. Pre-treatment blood samples from HNSCC patients were prospectively recruited through the Accelerated2 sample collection platform. RESULTS: In cell line spiking experiments, non-fixative EDTA blood collection tubes (BCTs) enriched with Parsortix demonstrated a mean capture rate of 53.5%, across a range of 9-129 cells/ml spiked concentrations (n=13). Transfix fixation BCTs demonstrated significantly improved capture rates of spiked cells at 0hr, 24hr and 72hr timepoints, when compared to EDTA BCTs. Parsortix enrichment significantly altered the gene expression of unfixed cells, causing downregulation of genes associated with RNA and ribosomal/protein processing genes and upregulation of genes associated with oxidative stress and cell injury/apoptosis. Using immunofluorescence microscopy, CTCs were identified from HNSCC patient samples (enriched with Parsortix) with epithelial (EpCAM expression) and epithelial-mesenchymal transition (EMT, EpCAM and N-cadherin expression) characteristics. In a cohort of 20 patients, flow cytometry characterisation of the above markers was able to quantify and characterise CTCs. Sixty-five percent (13/20) demonstrated CTCs, at a mean count of 4 CTCs/ml (range 2-11.2 CTCs/ml). The presence of CTCs correlated with advanced stage of disease (p=0.0121), but not T or N stage. Neither the presence of epithelial nor EMT CTC sub-groups correlated with progression-free or overall survival. A mesenchymal gene expression profile in patient matched primary tumour tissue did not positively correlate with CTC EMT expression (p=0.347). Mass cytometry analysis identified CTCs in 11 of 12 patient blood samples, with considerable CTC heterogeneity. Novel CTC sub-groups, common between patient samples, were defined based upon target marker expression patterns - for example EMT CTCs expressing proliferative markers and decreased immune-checkpoint markers. Mass cytometry outperformed bulk quantitative gene expression profiling of Parsortix enriched samples from the same cohort – both to identify the presence of CTCs and phenotypic sub-groups. A mesenchymal gene expression profile in primary tumour tissue was significantly associated with an increased proportion of proliferative and immune-checkpoint high CTCs. DISCUSSION: We demonstrate for the first time that the combination of Parsortix microfluidic enrichment and mass cytometry can be successfully used to provide multiparameter single-CTC characterisation of extracellular, intracellular and activated signalling proteins – a depth of information beyond that available from bulk CTC gene expression profiling. Even in a small cohort pilot study, we highlight an interesting trend that primary tumours with mesenchymal gene expression profiles exhibit increased levels of proliferative and immune-checkpoint high CTCs. The heterogeneity of CTC EMT and phenotypic subgroups and their relationship to immune-checkpoint markers is notable – providing both an avenue for therapeutic targeting, but also a means of therapeutic escape in ‘immune low’ CTCs. We lay a platform for future proteomic based single-CTC studies in larger cohorts

Cancer Biomarker Research and Personalized Medicine

Biomarkers are measures of a biological state. The treatment of individual patients based on particular factors, such as biomarkers, distinguishes standard, generalized treatment plans from personalized medicine. Even though personalized medicine is applicable to most branches of medicine, the field of oncology is perhaps where it is most easily employed. Cancer is a heterogeneous disease; although patients may be diagnosed histologically with the same cancer type, their tumors can comprise varying tumor microenvironments and molecular characteristics that can impact treatment response and prognosis. There has been a major drive over the past decade to try and realize personalized cancer medicine through the discovery and use of disease-specific biomarkers. This book, entitled “Cancer Biomarker Research and Personalized Medicine”, encompasses 22 publications from colleagues working on a diverse range of cancers, including prostate, breast, ovarian, head and neck, liver, gastric, bladder, colorectal, and kidney. The biomarkers assessed in these studies include genes, intracellular or secreted proteins, exosomes, DNA, RNA, miRNA, circulating tumor cells, circulating immune cells, in addition to radiomic features