Drug sensitivity profiling of 3D tumor tissue cultures in the pediatric precision oncology program INFORM

The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75-78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET, and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs

Drug sensitivity profiling of 3D tumor tissue cultures in the pediatric precision oncology program INFORM

The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75–78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET, and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs

Drug sensitivity profiling of 3D tumor tissue cultures in the pediatric precision oncology program INFORM

The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75-78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET, and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs.Peer reviewe

Molecular biomarkers in pediatric ependymoma

Ependymome zählen zu den häufigsten malignen Gehirntumoren im Kindesalter und machen insgesamt etwa 5-10% aller Gehirntumore in dieser Altersgruppe aus. Moderne molekularbiologische Technologien haben es ermöglicht die zugrundeliegende Biologie zu erforschen und somit die klinisch beobachteten Unterschiede, wie zum Beispiel die höhere Aggressivität von Ependymomen bei Kindern im Vergleich zu Erwachsenen, zu erklären. Trotz dieses Fortschritts fehlt es aber immer noch an verlässlichen Biomarkern um insbesondere Ependymome höherer Aggressivität innerhalb der pädiatrischen Ependymome zu identifizieren. Ziel dieser Dissertation war es deshalb neue molekulare Biomarker hinsichtlich ihrer klinischen sowie biologischen Relevanz zu untersuchen. Zu diesem Zweck wurde eine Kohorte von pädiatrischen Ependymomen für die Evaluation von neuen Biomarkern zusammengestellt und insbesondere hinsichtlich der klinischen Daten wie Therapie und Langzeitkontrolle detailliert charakterisiert. Im Rahmen unserer Studie wurde ein Zugewinn am langen Arm von Chromosom 1 (1q gain) als robuster Biomarker für aggressive Ependymome der hinteren Schädelgrube bestätigt. Zudem konnten wir belegen, dass diese Veränderung mit der Reaktivierung von Telomerase assoziiert ist, was eine zellbiologische Erklärung für das biologische Verhalten dieser Tumore bedeuten könnte. Außerdem stellte sich die Messung der Telomerase Aktivität als vielversprechender Biomarker innerhalb der „Posterior Fossa Group A“ Ependymome heraus. Darauffolgende umfassende Analysen unterschiedlicher Ependymomsubtypen zeigten, dass Telomerasereaktivierung ein zentrales Charakteristikum von aggressiven, nicht aber benignen Subgruppen darstellt und dass diesem Unterschied vermutlich eine differentielle Methylierung des Promoters des humanen Telomerase Reverse-Transkriptase (hTERT) Gens zugrunde liegt. Zuletzt konnten wir durch einen Vergleich von DNA Methylierungs- und Genexpressionsprofilen von Ependymomen mit anderen Tumorgruppen auch bestätigen, dass diese in seltenen Fällen auch anderen molekularen Tumortypen wie zum Beispiel „Central nervous system high-grade neuroepithelial tumor with BCL6-corepressor alteration“ (CNS HGNET-BCOR) zugeordnet werden können. Zusammenfassend konnte durch diese Arbeit der 1q gain als Biomarker bestätigt und die Telomerase Aktivität als neuer vielversprechender Marker in pädiatrischen Ependymomen evaluiert werden. Basierend auf diesen Ergebnissen sollten molekulare Analysen als Basis für zukünftige Diagnostik und Therapieentscheidungen herangezogen werden.Ependymomas are tumors of the central nervous system (CNS) representing the third most common malignant CNS tumor entity in children. Novel molecular high-throughput technologies have provided a basis to biologically explain some of the clinical observations such as age- or anatomy dependent differences in the clinical course of the disease. Nevertheless, also tumors within anatomic compartments or age groups may differ in terms of clinical outcome and biological behavior. Therefore, reliable biomarkers for meaningful patient stratification are urgently needed in order to improve the treatment of patients suffering from these highly aggressive tumor type. To address this question we compiled a retrospective study cohort of pediatric ependymomas treated at the Medical University of Vienna. Owing to the high importance of gross total resection and radiotherapy as well as the possibility of very late tumor recurrence in ependymoma, particular attention was paid to the collection of detailed clinical information including treatment as well as long-term follow-up data. Our analyses confirmed gain of chromosome 1q as robust prognosticator of inferior survival in posterior fossa ependymoma. Subsequent analyses revealed that 1q gain is associated with telomerase reactivation thereby providing a potential cell-biological reason for the higher aggressiveness of this subgroup. Furthermore, telomerase reactivation tested by the telomerase repeat amplification protocol (TRAP) proved to be a highly significant parameter for detection of tumors with dismal prognosis within the posterior fossa group A (PF-ENP-A) subgroup. More generally, we could even demonstrate that the presence of telomerase reactivation represents a hallmark characteristic of aggressive ependymoma subgroups but not more benign subtypes across anatomic compartments. In silico analysis of methylation patterns within a large set of ependymomas showed that this central difference seems to be based on distinct DNA methylation patterns within certain regions of the human telomerase reverse transcriptase (hTERT) promoter. Last, comparison of DNA methylation as well as gene-expression patterns of ependymoma to other tumor types uncovered that some rare cases might even fall into other molecularly defined tumor types such as CNS high-grade neuroepithelial tumor with BCL6-corepressor alteration (CNS HGNET-BCOR). Taken together, we confirm gain of chromosome 1q and present TRAP as promising biomarkers for pediatric ependymoma and further reinforce the central role of molecular diagnostics as ground for future clinical trials and decision making.submitted by Johannes GojoAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2018OeBB(VLID)257758

Proof-of-Concept for Liquid Biopsy Disease Monitoring of <i>MYC</i>-Amplified Group 3 Medulloblastoma by Droplet Digital PCR

Background: Liquid biopsy diagnostic methods are an emerging complementary tool to imaging and pathology techniques across various cancer types. However, there is still no established method for the detection of molecular alterations and disease monitoring in MB, the most common malignant CNS tumor in the pediatric population. In the presented study, we investigated droplet digital polymerase chain reaction (ddPCR) as a highly sensitive method for the detection of MYC amplification in bodily fluids of group 3 MB patients. Methods: We identified a cohort of five MYC-amplified MBs by methylation array and FISH. Predesigned and wet-lab validated probes for ddPCR were used to establish the detection method and were validated in two MYC-amplified MB cell lines as well as tumor tissue of the MYC-amplified cohort. Finally, a total of 49 longitudinal CSF samples were analyzed at multiple timepoints during the course of the disease. Results: Detection of MYC amplification by ddPCR in CSF showed a sensitivity and specificity of 90% and 100%, respectively. We observed a steep increase in amplification rate (AR) at disease progression in 3/5 cases. ddPCR was proven to be more sensitive than cytology for the detection of residual disease. In contrast to CSF, MYC amplification was not detectable by ddPCR in blood samples. Conclusions: ddPCR proves to be a sensitive and specific method for the detection of MYC amplification in the CSF of MB patients. These results warrant implementation of liquid biopsy in future prospective clinical trials to validate the potential for improved diagnosis, disease staging and monitoring

Prospective Evaluation of Kidney Function in Long-Term Survivors of Pediatric CNS Tumors

Purpose: Numerous acute effects of chemotherapeutics on kidney function are well described. However, data on the long-term effects of chemotherapy in the growing population of childhood central nervous system (CNS) tumor survivors is limited. We aimed to evaluate the kidney function of a cohort of long-term CNS tumor survivors treated with different standard chemotherapeutic regimens. Methods: Patients treated for a CNS tumor were prospectively evaluated up to 12 years after completion of their therapy. Examination of kidney function was performed during routine follow-up visits. Blood pressure and blood and urine parameters were analyzed for kidney function evaluation. Glomerular function was assessed by calculating the estimated glomerular filtration rate (eGFR), tubular functions were analyzed by measuring serum electrolytes, bicarbonate and phosphate reabsorption, and proteinuria was assessed by calculating the protein/creatinine ratio and phosphate reabsorption. Results: None of the 65 patients evaluated suffered from clinically relevant kidney impairment (eGFR &lt; 90 mL/min/L, 73 m2). There was no association between chemotherapy dose and eGFR. Only two patients showed mild signs of tubulopathy and 11 patients were diagnosed with elevated blood pressure. Conclusion: With adequate supportive measures, such as sufficient hydration according to chemotherapy protocol guidelines, as well as avoidance or close monitoring of additional nephrotoxic medication, impaired kidney function is rare in CNS tumor survivors treated with standard chemotherapy. Nonetheless, long-term follow-up is essential for early detection of mild impairment of kidney function

Asking those who know their needs best: A framework for active engagement and involvement of childhood cancer survivors and parents in the process of psychosocial research—A workshop report

Abstract Background Patient and public involvement and engagement (PPIE) in healthcare research is crucial for effectively addressing patients' needs and setting appropriate research priorities. However, there is a lack of awareness and adequate methods for practicing PPIE, especially for vulnerable groups like childhood cancer survivors. Aims This project aimed to develop and evaluate engagement methods to actively involve pediatric oncological patients, survivors, and their caregivers in developing relevant research questions and practical study designs. Methods and Results An interdisciplinary working group recruited n = 16 childhood cancer survivors and their caregivers to work through the entire process of developing a research question and a practicable study design. A systematic literature review was conducted to gather adequate PPIE methods which were then applied and evaluated in a series of three workshop modules, each lasting 1.5 days. The applied methods were continuously evaluated, while a monitoring group oversaw the project and continuously developed and adapted additional methods. The participants rated the different methods with varying scores. Over the workshop series, the participants successfully developed a research question, devised an intervention, and designed a study to evaluate their project. They also reported increased expertise in PPIE and research knowledge compared to the baseline. The project resulted in a practical toolbox for future research, encompassing the final workshop structure, evaluated methods and materials, guiding principles, and general recommendations. Conclusion These findings demonstrate that with a diverse set of effective methods and flexible support, actively involving patients, survivors, and caregivers can uncover patients' unmet disease‐related needs and generate practical solutions apt for scientific evaluation. The resulting toolbox, filled with evaluated and adaptable methods (workbook, Supplement 1 and 2), equips future scientists with the necessary resources to successfully perform PPIE in the development of health care research projects that effectively integrate patients' perspectives and address actual cancer‐related needs. This integration of PPIE practices has the potential to enhance the quality and relevance of health research and care, as well as to increase patient empowerment leading to sustainable improvements in patients' quality of life